Methods for generating hypermutable yeast

ABSTRACT

Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be be rendered genetically stable by restoring the mismatch repair system to proper functionality.

This application claims the benefit of provisional application Ser. No.60/184,336 filed Feb, 23, 2000.

FIELD OF THE INVENTION

The invention is related to the area of mismatch repair genes. Inparticular it is related to the field of in situ mutagenesis of singlecelled organisms.

BACKGROUND OF THE INVENTION

Within the past four years, the genetic cause of the HereditaryNonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynchsyndrome II, has been ascertained for the majority of kindred's affectedwith the disease (Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N.C., Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A.,Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Vogelstein, B., andKinzler, K. W. 1996. Analysis of mismatch repair genes in hereditarynon-polyposis colorectal cancer patients. Nat. Med. 2:169-174). Themolecular basis of HNPCC involves genetic instability resulting fromdefective mismatch repair (MMR). To date, six genes have been identifiedin humans that encode for proteins and appear to participate in the MMRprocess, including the mutS homologs GTBP, hMSH2, and hMSH3 and the mutLhomologs hALH1, hPMS1, and hPMS2 (Bronner, C. E., Baker, S. M.,Morrison, P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M.,Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., Bollag, R. J.,Godwin, A., R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R.,and Liskay, R. M. 1994. Mutation in the DNA mismatch repair genehomologue hMLH1 is associated with hereditary non-polyposis coloncancer. Nature 368:258-261; Fishel, R., Lescoe, M., Rao, M. R. S.,Copeland, N. J., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R.1993. The human mutator gene homolog MSH2 and its association withhereditary nonpolyposis colon cancer. Cell 7:1027-1038; Leach, F. S.,Nicolaides, N. C, Papadopoulos, N., Liu, B., Jen, J., Parsons, R.,Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan,X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J.,Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach R.J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, J. A.,Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P.,Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., andVogelstein, B. 1993. Mutations of a mutS homolog in hereditarynon-polyposis colorectal cancer. Cell 75:1215-1225; Nicolaides, N. C.,Papadopoulos, N., Liu, B., Wei, Y. F., Carter, K. C., Ruben, S. M.,Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M.,Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R., Petersen,G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K. W. 1994.Mutations of two PMS homologs in hereditary nonpolyposis colon cancer.Nature 371: 75-80; Nicolaides, N. C., Palombo, F., Kinzler, K. W.,Vogelstein, B., and Jiricny, J. 1996. Molecular cloning of theN-terminus of GTBP. Genomics 31:395-397; Palombo, F., Hughes, M.,Jiricny, J., Truong, O., Hsuan, J. 1994. Mismatch repair and cancer.Nature 36:417; Palombo, F., Gallinari, P., laccarino, I., Lettieri, T.,Hughes, M. A., Truong, O., Hsuan, J. J., and Jiricny, J. 1995. GTBP, a160-kilodalton protein essential for mismatch-binding activity in humancells. Science 268:1912-1914; Papadopoulos, N., Nicolaides, N. C., Wei,Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A.,Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop,M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein,B., and Kinzler, K. W. 1994. Mutation of a mutL homolog is associatedwith hereditary colon cancer. Science 263:1625-1629). Germline mutationsin four of these genes (hMSH2, hMLH1, hPMS1, and hPMS2) have beenidentified in HNPCC kindred's (Bronner, C. E., Baker, S. M., Morrison,P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C.,Lipford, J., Lindblom, A., Tannergard, P., Bollag, R. J., Godwin, A.,R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay,R. M. 1994. Mutation in the DNA mismatch repair gene homologue hMLH1 isassociated with hereditary non-polyposis colon cancer. Nature368:258-261; Leach, F. S., Nicolaides, N. C, Papadopoulos, N., Liu, B.,Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A.,Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W.,Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd,S., Lewis, T., Leach R. J., Naylor, S. L., Weissenbach, J., Mecklin, J.P., Jarvinen, J. A., Petersen, G. M., Hamilton, S. R., Green, J., Jass,J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler,K. W., and Vogelstein, B. 1993. Mutations of a mutS homolog inhereditary non-polyposis colorectal cancer. Cell 75:1215-1225; Liu, B.,Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson,P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de la Chapelle,A., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. 1996. Analysisof mismatch repair genes in hereditary non-polyposis colorectal cancerpatients. Nat. Med. 2:169-174; Nicolaides, N. C., Papadopoulos, N., Liu,B., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W.A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J.,Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A.,Vogelstein, B., and kinzler, K. W. 1994. Mutations of two PMS homologsin hereditary nonpolyposis colon cancer. Nature 371: 75-80;Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Carter, K. C., Ruben,S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C.M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R.,Petersen, G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K. W.1994. Mutation of a mutL homolog is associated with hereditary coloncancer. Science 263:1625-1629). Though the mutator defect that arisesfrom the MMR deficiency can affect any DNA sequence, microsatellitesequences are particularly sensitive to MMR abnormalities (Modrich, P.1994. Mismatch repair, genetic stability, and cancer. Science266:1959-1960). Microsatellite instability (MI) is therefore a usefulindicator of defective MMR. In addition to its occurrence in virtuallyall tumors arising in HNPCC patients, MI is found in a small fraction ofsporadic tumors with distinctive molecular and phenotypic properties(Perucho, M. 1996. Cancer of the microsattelite mutator phenotype. BiolChem. 377:675-684).

HNPCC is inherited in an autosomal dominant fashion, so that the normalcells of affected family members contain one mutant allele of therelevant MMR gene (inherited from an affected parent) and one wild-typeallele (inherited from the unaffected parent). During the early stagesof tumor development, however, the wild-type allele is inactivatedthrough a somatic mutation, leaving the cell with no functional MMR geneand resulting in a profound defect in MMR activity. Because a somaticmutation in addition to a germ-line mutation is required to generatedefective MMR in the tumor cells, this mechanism is generally referredto as one involving two hits, analogous to the biallelic inactivation oftumor suppressor genes that initiate other hereditary cancers (Leach, F.S., Nicolaides, N. C, Papadopoulos, N., Liu, B., Jen, J., Parsons, R.,Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan,X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J.,Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach R.J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, J. A.,Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P.,Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., andVogelstein, B. 1993. Mutations of a mutS homolog in hereditarynon-polyposis colorectal cancer. Cell 75:1215-1225; Liu, B., Parsons,R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson, P., Jass,J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de la Chapelle, A.,Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. 1996. Analysis ofmismatch repair genes in hereditary non-polyposis colorectal cancerpatients. Nat. Med. 2:169-174; Parsons, R., Li, G. M., Longley, M. J.,Fang, W. H., Papadopolous, N., Jen, J., de la Chapelle, A., Kinzler, K.W., Vogelstein, B., and Modrich, P. 1993. Hypermutability and mismatchrepair deficiency in RER⁺ tumor cells. Cell 75:1227-1236). In line withthis two-hit mechanism, the non-neoplastic cells of HNPCC patientsgenerally retain near normal levels of MMR activity due to the presenceof the wild-type allele.

The ability to alter the signal transduction pathways by manipulation ofa gene products function, either by over-expression of the wild typeprotein or a fragment thereof, or by introduction of mutations intospecific protein domains of the protein, the so-called dominant-negativeinhibitory mutant, were described over a decade in the yeast systemSaccharomyces cerevisiae by Herskowitz (Nature 329(6136):219-222, 1987).It has been demonstrated that over-expression of wild type gene productscan result in a similar, dominant-negative inhibitory phenotype due mostlikely to the “saturating-out” of a factor, such as a protein, that ispresent at low levels and necessary for activity; removal of the proteinby binding to a high level of its cognate partner results in the samenet effect, leading to inactivation of the protein and the associatedsignal transduction pathway. Recently, work done by Nicolaides et. al.(Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998.A naturally occurring hPMS2 mutation can confer a dominant negativemutator phenotype. Mol Cell Biol 18:1635-1641) has demonstrated theutility of introducing dominant negative inhibitory mismatch repairmutants into mammalian cells to confer global DNA hypermutability. Theability to manipulate the MMR process and therefore increase themutability of the target host genome at will, in this example amammalian cell, allows for the generation of innovative cell subtypes orvariants of the original wild type cells. These variants can be placedunder a specified, desired selective process, the result of which is anovel organism that expresses an altered biological molecule(s) and hasa new trait. The concept of creating and introducing dominant negativealleles of a gene, including the MMR alleles, in bacterial cells hasbeen documented to result in genetically altered prokaryotic mismatchrepair genes (Aronshtam A, Marinus M G. 1996. Dominant negative mutatormutations in the mutL gene of Escherichia coli. Nucleic Acids Res24:2498-2504; Wu T H, Marinus M G. 1994. Dominant negative mutatormutations in the mutS gene of Escherichia coli. J Bacteriol176:5393-400; Brosh R M Jr, Matson S W. 1995. Mutations in motif II ofEscherichia coli DNA helicase II render the enzyme nonfunctional in bothmismatch repair and excision repair with differential effects on theunwinding reaction. J Bacteriol 177:5612-5621). Furthermore, altered MMRactivity has been demonstrated when MMR genes from different speciesincluding yeast, mammalian cells, and plants are over-expressed (Fishel,R., Lescoe, M., Rao, M. R. S., Copeland, N. J., Jenkins, N. A., Garber,J., Kane, M., and Kolodner, R. 1993. The human mutator gene homolog MSH2and its association with hereditary nonpolyposis colon cancer. Cell7:1027-1038; Studamire B, Quach T, Alani, E. 1998. Saccharomycescerevisiae Msh2p and Msh6p ATPase activities are both required duringmismatch repair. Mol Cell Biol 18:7590-7601; Alani E, Sokolsky T,Studamire B, Miret J J, Lahue R S. 1997. Genetic and biochemicalanalysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunitinteractions in mismatch base pair recognition. Mol Cell Biol17:2436-2447; Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis AD, Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatchrepair gene associated with mammalian microsatellite instability. Nat.Genet. 24:27-35).

There is a continuing need in the art for methods of geneticallymanipulating useful strains of yeast to increase their performancecharacteristics and abilities.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forrendering yeast cells hypermutable.

It is another object of the invention to provide hypermutable yeastcells.

It is a further object of the invention to provide a method of mutatinga gene of interest in a yeast.

It is yet another object of the present invention to provide a method toproduce yeast that are hypermutable.

It is an object of the invention to provide a method to restore normalmismatch repair activity to hypermutable cells following strainselection.

These and other objects of the invention are provided by one or more ofthe following embodiments. In one embodiment a method is provided formaking a hypermutable yeast. A polynucleotide comprising a dominantnegative allele of a mismatch repair gene is introduced into a yeastcell. The cell thus becomes hypermutable.

According to another embodiment a homogeneous composition of cultured,hypermutable yeast cells is provided. The yeast cells comprise adominant negative allele of a mismatch repair gene.

According to still another embodiment of the invention a method isprovided for generating a mutation in a gene of interest. A yeast cellculture comprising the gene of interest and a dominant negative alleleof a mismatch repair gene is cultivated. The yeast cell is hypermutable.Cells of the culture are tested to determine whether the gene ofinterest harbors a mutation.

In yet another embodiment of the invention a method is provided forgenerating a mutation in a gene of interest. A yeast cell comprising thegene of interest and a polynucleotide encoding a dominant negativeallele of a mismatch repair gene is grown to create a population ofmutated, hypermutable yeast cells. The population of mutated,hypermutable yeast cells is cultivated under trait selection conditions.Yeast cells which grow under trait selection conditions are tested todetermine whether the gene of interest harbors a mutation.

Also provided by the present invention is a method for generatingenhanced hypermutable yeast. A yeast cell is exposed to a mutagen. Theyeast cell is defective in mismatch repair (MMR) due to the presence ofa dominant negative allele of at least one MMR gene. An enhanced rate ofmutation of the yeast cell is achieved due to the exposure to themutagen.

According to still another aspect of the invention a method is providedfor generating mismatch repair (MMR)-proficient yeast with new outputtraits. A yeast cell comprising a gene of interest and a polynucleotideencoding a dominant negative allele of a mismatch repair gene is grownto create a population of mutated, hypermutable yeast cells. Thepopulation of mutated, hypermutable yeast cells is cultivated undertrait selection conditions. The yeast cells which grow under traitselection conditions are tested to determine whether the gene ofinterest harbors a mutation. Normal mismatch repair activity is restoredto the yeast cells.

These and other embodiments of the invention provide the art withmethods that can generate enhanced mutability in yeast as well asproviding single-celled eukaryotic organisms harboring potentiallyuseful mutations to generate novel output traits for commercialapplications.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that hypermutable yeast canbe made by altering the activity of endogenous mismatch repair activityof host cells. Dominant negative alleles of mismatch repair genes, whenintroduced and expressed in yeast, increase the rate of spontaneousmutations by reducing the effectiveness of endogenous mismatchrepair-mediated DNA repair activity, thereby rendering the yeast highlysusceptible to genetic alterations, i.e., hypermutable. Hypermutableyeast can then be utilized to screen for mutations in a gene or a set ofgenes in variant siblings that exhibit an output trait(s) not found inthe wild-type cells.

The process of mismatch repair, also called mismatch proofreading, is anevolutionarily highly conserved process that is carried out by proteincomplexes described in cells as disparate as prokaryotic cells such asbacteria to more complex mammalian cells (Modrich, P. 1994. Mismatchrepair, genetic stability, and cancer. Science 266:1959-1960; Parsons,R., Li, G. M., Longley, M., Modrich, P., Liu, B., Berk, T., Hamilton, S.R., Kinzler, K. W., and Vogelstein, B. 1995. Mismatch repair deficiencyin phenotypically normal human cells. Science 268:738-740; Perucho, M.1996. Cancer of the microsattelite mutator phenotype. Biol Chem.377:675-684). A mismatch repair gene is a gene that encodes one of theproteins of such a mismatch repair complex. Although not wanting to bebound by any particular theory of mechanism of action, a mismatch repaircomplex is believed to detect distortions of the DNA helix resultingfrom non-complementary pairing of nucleotide bases. Thenon-complementary base on the newer DNA strand is excised, and theexcised base is replaced with the appropriate base that is complementaryto the older DNA strand. In this way, cells eliminate many mutationsthat occur as a result of mistakes in DNA replication, resulting ingenetic stability of the sibling cells derived from the parental cell.

Some wild type alleles as well as dominant negative alleles cause amismatch repair defective phenotype even in the presence of a wild-typeallele in the same cell. An example of a dominant negative allele of amismatch repair gene is the human gene hPMS2-134, which carries atruncation mutation at codon 134 (Parsons, R., Li, G. M., Longley, M.,Modrich, P., Liu, B., Berk, T., Hamilton, S. R., Kinzler, K. W., andVogelstein, B. 1995. Mismatch repair deficiency in phenotypically normalhuman cells. Science 268:738-740; Nicolaides N C, Littman S J, ModrichP, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutationcan confer a dominant negative mutator phenotype. Mol Cell Biol18:1635-1641). The mutation causes the product of this gene toabnormally terminate at the position of the 134th amino acid, resultingin a shortened polypeptide containing the N-terminal 133 amino acids.Such a mutation causes an increase in the rate of mutations, whichaccumulate in cells after DNA replication. Expression of a dominantnegative allele of a mismatch repair gene results in impairment ofmismatch repair activity, even in the presence of the wild-type allele.Any mismatch repair allele, which produces such effect, can be used inthis invention, whether it is wild-type or altered, whether it derivesfrom mammalian, yeast, fungal, amphibian, insect, plant, or bacteria. Inaddition, the use of over-expressed wild type MMR gene alleles fromhuman, mouse, plants, and yeast in bacteria has been shown to cause adominant negative effect on the bacterial hosts MMR activity (AronshtamA, Marinus M G. 1996. Dominant negative mutator mutations in the mutLgene of Escherichia coli. Nucleic Acids Res 24:2498-2504; Wu T H,Marinus M G. 1994. Dominant negative mutator mutations in the mutS geneof Escherichia coli. J Bacteriol 176:5393-400; Brosh R M Jr, Matson S W.1995. Mutations in motif II of Escherichia coli DNA helicase II renderthe enzyme nonfunctional in both mismatch repair and excision repairwith differential effects on the unwinding reaction. J Bacteriol177:5612-5621; Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, BaxevanisA D, Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatchrepair gene associated with mammalian microsatellite instability. NatGenet 24:27-35). This suggests that perturbation of the multi-componentMMR protein complex can be accomplished by introduction of MMRcomponents from other species into yeast.

Dominant negative alleles of a mismatch repair gene can be obtained fromthe cells of humans, animals, yeast, bacteria, plants or otherorganisms. Screening cells for defective mismatch repair activity canidentify such alleles. Mismatch repair genes may be mutant or wild type.Yeast host MMR may be mutated or not. The term yeast used in thisapplication comprises any organism from the eukaryotic kingdom,including but not limited to Saccharomyces sp., Pichia sp.,Schizosaccharomyces sp., Kluyveromyces sp., and other fungi (Gellissen,G. and Hollenberg, C P. Gene 190(1):87-97, 1997). These organisms can beexposed to chemical mutagens or radiation, for example, and can bescreened for defective mismatch repair. Genomic DNA, cDNA, mRNA, orprotein from any cell encoding a mismatch repair protein can be analyzedfor variations from the wild-type sequence. Dominant negative alleles ofa mismatch repair gene can also be created artificially, for example, byproducing variants of the hPMS2-134 allele or other mismatch repairgenes (Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B1998. A naturally occurring hPMS2 mutation can confer a dominantnegative mutator phenotype. Mol Cell Biol 18:1635-1641). Varioustechniques of site-directed mutagenesis can be used. The suitability ofsuch alleles, whether natural or artificial, for use in generatinghypermutable yeast can be evaluated by testing the mismatch repairactivity (using methods described in Nicolaides N C, Littman S J,Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2mutation can confer a dominant negative mutator phenotype. Mol Cell Biol18:1635-1641) caused by the allele in the presence of one or morewild-type alleles to determine if it is a dominant negative allele.

A yeast that over-expresses a wild type mismatch repair allele or adominant negative allele of a mismatch repair gene will becomehypermutable. This means that the spontaneous mutation rate of suchyeast is elevated compared to yeast without such alleles. The degree ofelevation of the spontaneous mutation rate can be at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal yeast as measured as a function of yeastdoubling/hour.

According to one aspect of the invention, a polynucleotide encodingeither a wild type or a dominant negative form of a mismatch repairprotein is introduced into yeast. The gene can be any dominant negativeallele encoding a protein which is part of a mismatch repair complex,for example, mutS, mutL, mutH, or mutY homologs of the bacterial, yeast,plant or mammalian genes (Modrich, P. 1994. Mismatch repair, geneticstability, and cancer. Science 266:1959-1960; Prolla, T. A, Pang, Q.,Alani, E., Kolodner, R. A., and Liskay, R. M. 1994. MLH1, PMS1, and MSH2Interaction during the initiation of DNA mismatch repair in yeast.Science 264:1091-1093). The gene may also be a PMSR gene such as hPMSR,PMSR2 and PMSR3, or a PMSL gene. The gene may also be MSH6. The dominantnegative allele can be naturally occurring or made in the laboratory.The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or achemically synthesized polynucleotide or polypeptide. The molecule canbe introduced into the cell by transformation, electroporation, mating,particle bombardment, or other method described in the literature.

Transformation is used herein as any process whereby a polynucleotide orpolypeptide is introduced into a cell. The process of transformation canbe carried out in a yeast culture using a suspension of cells. The yeastcan be any type classified under the eukayotic kingdom as byinternational convention.

In general, transformation will be carried out using a suspension ofcells but other methods can also be employed as long as a sufficientfraction of the treated cells incorporate the polynucleotide orpolypeptide so as to allow transfected cells to be grown and utilized.The protein product of the polynucleotide may be transiently or stablyexpressed in the cell. Techniques for transformation are well known tothose skilled in the art. Available techniques to introduce apolynucleotide or polypeptide into a yeast cell include but are notlimited to electroporation, viral transduction, cell fusion, the use ofspheroplasts or chemically competent cells (e.g., calcium chloride), andpackaging of the polynucleotide together with lipid for fusion with thecells of interest. Once a cell has been transformed with the mismatchrepair gene or protein, the cell can be propagated and manipulated ineither liquid culture or on a solid agar matrix, such as a petri dish.If the transfected cell is stable, the gene will be expressed at aconsistent level for many cell generations, and a stable, hypermutableyeast strain results.

An isolated yeast cell can be obtained from a yeast culture bychemically selecting strains using antibiotic selection of an expressionvector. If the yeast cell is derived from a single cell, it is definedas a clone. Techniques for single-cell cloning of microorganisms such asyeast are well known in the art.

A polynucleotide encoding a dominant negative form of a mismatch repairprotein can be introduced into the genome of yeast or propagated on anextra-chromosomal plasmid, such as the 2-micron plasmid. Selection ofclones harboring a mismatch repair gene expression vector can beaccomplished by plating cells on synthetic complete medium lacking theappropriate amino acid or other essential nutrient as described (J. C.Schneider and L. Guarente, Methods in Enzymology 194:373,1991). Theyeast can be any species for which suitable techniques are available toproduce transgenic microorganisms, such as but not limited to generaincluding Saccharomyces, Schizosaccharomyces, Pichia, Hansenula,Kluyveromyces and others.

Any method for making transgenic yeast known in the art can be used.According to one process of producing a transgenic microorganism, thepolynucleotide is introduced into the yeast by one of the methods wellknown to those in the art. Next, the yeast culture is grown underconditions that select for cells in which the polynucleotide encodingthe mismatch repair gene is either incorporated into the host genome asa stable entity or propagated on a self-replicating extra-chromosomalplasmid, and the protein encoded by the polynucleotide fragmenttranscribed and subsequently translated into a functional protein withinthe cell. Once transgenic yeast is engineered to harbor the expressionconstruct, it is then propagated to generate and sustain a culture oftransgenic yeast indefinitely.

Once a stable, transgenic yeast cell has been engineered to express adefective mismatch repair (MMR) protein, the yeast can be cultivated tocreate novel mutations in one or more target gene(s) of interestharbored within the same yeast cell. A gene of interest can be any genenaturally possessed by the yeast or one introduced into the yeast hostby standard recombinant DNA techniques. The target gene(s) may be knownprior to the selection or unknown. One advantage of employing suchtransgenic yeast cells to induce mutations in resident orextra-chromosomal genes within the yeast is that it is unnecessary toexpose the cells to mutagenic insult, whether it is chemical orradiation, to produce a series of random gene alterations in the targetgene(s). This is due to the highly efficient nature and the spectrum ofnaturally occurring mutations that result as a consequence of thealtered mismatch repair process. However, it is possible to increase thespectrum and frequency of mutations by the concomitant use of eitherchemical and/or radiation together with MMR defective cells. The neteffect of the combination treatment is an increase in mutation rate inthe genetically altered yeast that are useful for producing new outputtraits. The rate of the combination treatment is higher than the rateusing only the MMR-defective cells or only the mutagen with wild-typeMMR cells.

MMR-defective yeast of the invention can be used in genetic screens forthe direct selection of variant sub-clones that exhibit new outputtraits with commercially desirable applications. This permits one tobypass the tedious and time consuming steps of gene identification,isolation and characterization.

Mutations can be detected by analyzing the internally and/or externallymutagenized yeast for alterations in its genotype and/or phenotype.Genes that produce altered phenotypes in MMR-defective microbial cellscan be discerned by any of a variety of molecular techniques well knownto those in the art. For example, the yeast genome can be isolated and alibrary of restriction fragments of the yeast genome can be cloned intoa plasmid vector. The library can be introduced into a “normal” cell andthe cells exhibiting the novel phenotype screened. A plasmid can beisolated from those normal cells that exhibit the novel phenotype andthe gene(s) characterized by DNA sequence analysis. Alternatively,differential messenger RNA screen can be employed utilizing driver andtester RNA (derived from wild type and novel mutant, respectively)followed by cloning the differential transcripts and characterizing themby standard molecular biology methods well known to those skilled in theart. Furthermore, if the mutant sought is encoded by anextra-chromosomal plasmid, then following co-expression of the dominantnegative MMR gene and the gene of interest, and following phenotypicselection, the plasmid can be isolated from mutant clones and analyzedby DNA sequence analysis using methods well known to those in the art.Phenotypic screening for output traits in MMR-defective mutants can beby biochemical activity and/or a readily observable phenotype of thealtered gene product. A mutant phenotype can also be detected byidentifying alterations in electrophoretic mobility, DNA binding in thecase of transcription factors, spectroscopic properties such as IR, CD,X-ray crystallography or high field NMR analysis, or other physical orstructural characteristics of a protein encoded by a mutant gene. It isalso possible to screen for altered novel function of a protein in situ,in isolated form, or in model systems. One can screen for alteration ofany property of the yeast associated with the function of the gene ofinterest, whether the gene is known prior to the selection or unknown.

The screening and selection methods discussed are meant to illustratethe potential means of obtaining novel mutants with commerciallyvaluable output traits, but they are not meant to limit the manypossible ways in which screening and selection can be carried out bythose of skill in the art.

Plasmid expression vectors that harbor a mismatch repair (MMR) geneinsert can be used in combination with a number of commerciallyavailable regulatory sequences to control both the temporal andquantitative biochemical expression level of the dominant negative MMRprotein. The regulatory sequences can be comprised of a promoter,enhancer or promoter/enhancer combination and can be inserted eitherupstream or downstream of the MMR gene to control the expression level.The regulatory sequences can be any of those well known to those in theart, including but not limited to the AOX1, GAP, GAL1, GAL10, PHO5, andPGK promoters harbored on high or low copy number extra-chromosomalexpression vectors or on constructs that are integrated into the genomevia homologous recombination. These types of regulatory systems havebeen disclosed in scientific publications and are familiar to thoseskilled in the art.

Once a microorganism with a novel, desired output trait of interest iscreated, the activity of the aberrant MMR activity is desirablyattenuated or eliminated by any means known in the art. These includebut are not limited to removing an inducer from the culture medium thatis responsible for promoter activation, curing a plasmid from atransformed yeast cell, and addition of chemicals, such as5-fluoro-orotic acid to “loop-out” the gene of interest.

In the case of an inducibly controlled dominant negative MMR allele,expression of the dominant negative MMR gene will be turned on (induced)to generate a population of hypermutable yeast cells with new outputtraits. Expression of the dominant negative MMR allele can be rapidlyturned off to reconstitute a genetically stable strain that displays anew output trait of commercial interest. The resulting yeast strain isnow useful as a stable strain that can be applied to various commercialapplications, depending upon the selection process placed upon it.

In cases where genetically deficient mismatch repair yeast [strains suchas but not limited to: EC2416 (mutS delta umuDC), and mutL or mutYstrains] are used to derive new output traits, transgenic constructs canbe used that express wild type mismatch repair genes sufficient tocomplement the genetic defect and therefore restore mismatch repairactivity of the host after trait selection [Grzesiuk, E. et.al.(Mutagenesis 13;127-132, 1998); Bridges, B. A., et.al. (EMBO J.16:3349-3356, 1997); LeClerc, J. E., Science 15:1208-1211, 1996);Jaworski, A. et.al. (Proc. Nati. Acad. Sci USA 92:11019-11023, 1995)].The resulting yeast is genetically stable and can be employed forvarious commercial applications.

The use of over-expression of foreign (exogenous, transgenic) mismatchrepair genes from human and yeast such as MSH2, MLH1, MLH3, etc. havebeen previously demonstrated to produce a dominant negative mutatorphenotype in yeast hosts (Shcherbakova, P. V., Hall, M. C., Lewis, M.S., Bennett, S. E., Martin, K. J., Bushel, P. R., Afshari, C. A., andKunkel, T. A. Mol. Cell Biol. 21(3):940-951; Studamire B, Quach T,Alani, E. 1998. Saccharomyces cerevisiae Msh2p and Msh6p ATPaseactivities are both required during mismatch repair. Mol Cell Biol18:7590-7601; Alani E, Sokolsky T, Studamire B, Miret J J, Lahue R S.1997. Genetic and biochemical analysis of Msh2p-Msh6p: role of ATPhydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pairrecognition. Mol Cell Biol 17:2436-2447; Lipkin S M, Wang V, Jacoby R,Banerjee-Basu S, Baxevanis A D, Lynch H T, Elliott R M, and Collins F S.2000. MLH3: a DNA mismatch repair gene associated with mammalianmicrosatellite instability. Nat Genet 24:27-35). In addition, the use ofyeast strains expressing prokaryotic dominant negative MMR genes as wellas hosts that have genomic defects in endogenous MMR proteins have alsobeen previously shown to result in a dominant negative mutator phenotype(Evans, E., Sugawara, N., Haber, J. E., and Alani, E. Mol. Cell.5(5):789-799, 2000; Aronshtam A, Marinus M G. 1996. Dominant negativemutator mutations in the mutL gene of Escherichia coli. Nucleic AcidsRes 24:2498-2504; Wu T H, Marinus M G. 1994. Dominant negative mutatormutations in the mutS gene of Escherichia coli. J Bacteriol176:5393-400; Brosh R M Jr, Matson S W. 1995. Mutations in motif II ofEscherichia coli DNA helicase II render the enzyme nonfunctional in bothmismatch repair and excision repair with differential effects on theunwinding reaction. J Bacteriol 177:5612-5621). However, the findingsdisclosed here teach the use of MMR genes, including the human PMSR2gene (Nicolaides, N. C., Carter, K. C., Shell, B. K., Papadopoulos, N.,Vogelstein, B., and Kinzler, K. W. 1995. Genomic organization of thehuman PMS2 gene family. Genomics 30:195-206), the related PMS134truncated MMR gene (Nicolaides N. C., Kinzler, K. W., and Vogelstein, B.1995. Analysis of the 5′ region of PMS2 reveals heterogenous transcriptsand a novel overlapping gene. Genomics 29:329-334), the plant mismatchrepair genes (U.S. patent application Ser. No. 09/749,601) and thosegenes that are homologous to the 134 N-terminal amino acids of the PMS2gene to create hypermutable yeast.

DNA mutagens can be used in combination with MMR defective yeast hoststo enhance the hypermutable production of genetic alterations. Thisfurther reduces MMR activity and is useful for generation ofmicroorganisms with commercially relevant output traits.

The ability to create hypermutable organisms using dominant negativealleles can be used to generate innovative yeast strains that displaynew output features useful for a variety of applications, including butnot limited to the manufacturing industry, for the generation of newbiochemicals, for detoxifying noxious chemicals, either by-products ofmanufacturing processes or those used as catalysts, as well as helpingin remediation of toxins present in the environment, including but notlimited to polychlorobenzenes (PCBs), heavy metals and otherenvironmental hazards. Novel yeast strains can be selected for enhancedactivity to either produce increased quantity or quality of a protein ornon-protein therapeutic molecule by means of biotransformation.Biotransformation is the enzymatic conversion of one chemicalintermediate to the next intermediate or product in a pathway or schemeby a microbe or an extract derived from the microbe. There are manyexamples of biotransformation in use for the commercial manufacturing ofimportant biological and chemical products, including penicillin G,erythromycin, and clavulanic acid. Organisms that are efficient atconversion of “raw” materials to advanced intermediates and/or finalproducts also can perform biotransformation (Beny, A. Trends Biotechnol.14(7):250-256). The ability to control DNA hypermutability in host yeaststrains using a dominant negative MMR (as described above) allows forthe generation of variant subtypes that can be selected for newphenotypes of commercial interest, including but not limited toorganisms that are toxin-resistant, have the capacity to degrade a toxinin situ or the ability to convert a molecule from an intermediate toeither an advanced intermediate or a final product. Other applicationsusing dominant negative MMR genes to produce genetic alteration of yeasthosts for new output traits include but are not limited to recombinantproduction strains that produce higher quantities of a recombinantpolypeptide as well as the use of altered endogenous genes that cantransform chemical or catalyze manufacturing downstream processes. Aregulatable dominant negative MMR phenotype can be used to produce ayeast strain with a commercially beneficial output trait. Using thisprocess, single-celled yeast cells expressing a dominant negative MMRcan be directly selected for the phenotype of interest. Once a selectedyeast with a specified output trait is isolated, the hypermutableactivity of the dominant negative MMR allele can be turned-off byseveral methods well known to those skilled in the art. For example, ifthe dominant-negative allele is expressed by an inducible promotersystem, the inducer can be removed or depleted. Such systems include butare not limited to promoters such as: lactose inducible GALi-GAL10promoter (M. Johnston and R. W. Davis, Mol. Cell Biol. 4:1440, 1984);the phosphate inducible PHO5 promoter (A. Miyanohara, A. Toh-e, C.Nosaki, F. Nosaki, F. Hamada, N. Ohtomo, and K. Matsubara. Proc. Nati.Acad. Sci. U.S.A. 80:1, 1983); the alcohol dehydrogenase I (ADH) and3-phosphoglycerate kinase (PGK) promoters, that are considered to beconstitutive but can be repressed/de-repressed when yeast cells aregrown in non-fermentable carbon sources such as but not limited tolactate (G. Ammerer, Methods in Enzymology 194:192, 1991; J. Mellor, M.J. Dobson, N. A. Roberts, M. F. Tuite, J. S. Emtage, S. White, D. A.Lowe, T. Patel, A. J. Kingsman, and S. M. Kingsman, Gene 24:563, 1982);S. Hahn and L. Guarente, Science 240:317, 1988); Alcohol oxidase (AOX)in Pichia vastoris (Tschopp, J F, Brust, P F, Cregg, J M, Stillman, C A,and Gingeras, T R. Nucleic Acids Res. 15(9):3859-76, 1987; and thethiamine repressible expression promoter nmt1 in Schizosaccharomycespombe (Moreno, M B, Duran, A., and Ribas, J C. Yeast 16(9):861-72,2000). Yeast cells can be transformed by any means known to thoseskilled in the art, including chemical transformation with LiC1 (Mount,R. C., Jordan, B. E., and Hadfield, C. Methods Mol. Biol.53:139-145,1996) and electroporation (Thompson, J R, Register, E.,Curotto, J., Kurtz, M. and Kelly, R. Yeast 14(6):565-71, 1998). Yeastcells that have been transformed with DNA can be selected for growth bya variety of methods, including but not restricted to selectable markers(URA3; Rose, M., Grisafi, P., and Botstein, D. Gene 29:113,1984; LEU2;A. Andreadis, Y., Hsu, M., Hermodson, G., Kohihaw, and P. Schimmel. J.Biol. Chem. 259:8059,1984; ARG4; G. Tschumper and J. Carbon. Gene10:157, 1980; and HIS3; K. Struhl, D. T. Stinchcomb, S., Scherer, and R.W. Davis Proc. Natl. Acad. Sci. U.S.A. 76:1035,1979) and drugs thatinhibit growth of yeast cells (tunicamycin, TUN; S. Hahn, J., Pinkham,R. Wei, R., Miller, and L. Guarente. Mol. Cell Biol. 8:655,1988).Recombinant DNA can be introduced into yeast as described above and theyeast vectors can be harbored within the yeast cell eitherextra-chromosomally or integrated into a specific locus.Extra-chromosomal based yeast expression vectors can be either high copybased (such as the 2-.mu.m vector Yep13; A. B. Rose and J. R. Broach,Methods in Enzymology 185:234,1991), low copy centromeric vectors thatcontain autonomously replicating sequences (ARS) such as YRp7 (M.Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235,1982) and wellas integration vectors that permit the gene of interest to be introducedinto specified locus within the host genome and propagated in a stablemaimer (R. J. Rothstein, Methods in Enzymology 101:202, 1991). Ectopicexpression of MMR genes in yeast can be attenuated or completelyeliminated at will by a variety of methods, including but not limited toremoval from the medium of the specific chemical inducer (e.g., depletegalactose that drives expression of the GAL10 promoter in Saccharomycescerevisiae or methanol that drives expression of the AOX1 promoter inPichia pastoris), extra-chromosomally replicating plasmids can be“cured” of expression plasmid by growth of cells under non-selectiveconditions (e.g. YEp13 harboring cells can be propagated in the presenceof leucine) and cells that have genes inserted into the genome can begrown with chemicals that force the inserted locus to “loop-out” (e.g.,integrants that have URA3 can be selected for loss of the inserted geneby growth of integrants on 5-fluoro-orotic acid (J. D. Boeke, F.LaCroute and G. R. Fink. Mol. Gen. Genet. 197:345-346,1984). Whether bywithdrawal of inducer or treatment of yeast cells with chemicals,removal of MMR expression results in the re-establishment of agenetically stable yeast cell-line. Thereafter, the lack of mutant MMRallows the endogenous, wild type MMR activity in the host cell tofunction normally to repair DNA. The newly generated mutant yeaststrains that exhibit novel, selected output traits are suitable for awide range of commercial processes or for gene/protein discovery toidentify new biomolecules that are involved in generating a particularoutput trait. While it has been documented that MMR deficiency can leadto as much as a 1000-fold increase in the endogenous DNA mutation rateof a host, there is no assurance that MMR deficiency alone will besufficient to alter every gene within the DNA of the host bacterium tocreate altered biochemicals with new activity(s). Therefore, the use ofchemical mutagens and their respective analogues such as ethidiumbromide, EMS, MNNG, MNu, Tamoxifen, 8-Hydroxyguanine, as well as otherssuch as those taught in: Khromov-Borisov, N. N., et al. (Mutat. Res. 430:55-74, 1999); Ohe, T., et al. (Mutat. Res. 429:189-199, 1999); Hour,T. C. et al. (Food Chem. Toxicol. 37:569-579, 1999); Hrelia, P., et al.(Chem. Biol. Interact. 118:99-111, 1999); Garganta, F., et al. (Environ.Mol. Mutagen. 33:75-85, 1999); Ukawa-Ishikawa S., et al. (Mutat. Res.412:99-107, 1998); the website having the URL address: www host server,ehs.utah.edu domain name, ohh directory, mutagens subdirectory, etc. canbe used to further enhance the spectrum of mutations and increase thelikelihood of obtaining alterations in one or more genes that can inturn generate host yeast with a desired new output trait(s). Mismatchrepair deficiency leads to hosts with an increased resistance totoxicity by chemicals with DNA damaging activity. This feature allowsfor the creation of additional genetically diverse hosts when mismatchdefective yeast are exposed to such agents, which would be otherwiseimpossible due to the toxic effects of such chemical mutagens [Colella,G., et al. (Br. J. Cancer 80:338-343, 1999); Moreland, N. J., et al.(Cancer Res. 59:2102-2106, 1999); Humbert, O., et. al. (Carcinogenesis20:205-214, 1999); Glaab, W. E., et al. (Mutat. Res. 398:197-207,1998)]. Moreover, mismatch repair is responsible for repairingchemically-induced DNA adducts, therefore blocking this process couldtheoretically increase the number, types, mutation rate and genomicalterations of a yeast1 [Rasmussen, L. J. et al. (Carcinogenesis17:2085-2088, 1996); Sledziewska-Gojska, E., et al. (Mutat. Res.383:31-37, 1997); and Janion, C. et al. (Mutat. Res. 210:15-22, 1989)].In addition to the chemicals listed above, other types of DNA mutagensinclude ionizing radiation and UV-irradiation, which is known to causeDNA mutagenesis in yeast, can also be used to potentially enhance thisprocess (Lee C C, Lin H K, Lin J K, 1994. A reverse mutagenicity assayfor alkylating agents based on a point mutation in the beta-lactamasegene at the active site serine codon. Mutagenesis 9:401-405; Vidal A,Abril N, Pueyo C. 1995. DNA repair by Ogt alkyltransferase influencesEMS mutational specificity. Carcinogenesis 16:817-821). These agents,which are extremely toxic to host cells and therefore result in adecrease in the actual pool size of altered yeast cells are moretolerated in MMR defective hosts and in turn permit an enriched spectrumand degree of genomic mutagenesis.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples that will be provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLES Example 1 Generation of Inducible MMR Dominant Negative AlleleVectors and Yeast Cells Harboring the Expression Vectors

Yeast expression constructs were prepared to determine if the human PMS2related gene (hPMSR2) (Nicolaides et al. Genomics 30(2):195-206) and thehuman PMS134 gene (Nicolaides N C, Littman S J, Modrich P, Kinzler K W,Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer adominant negative mutator phenotype. Mol Cell Biol 18:1635-1641) arecapable of inactivating the yeast MMR activity and thereby increase theoverall frequency of genomic hypermutation, a consequence of which isthe generation of variant sib cells with novel output traits followinghost selection. For these studies, a plasmid encoding the hPMS134 cDNAwas altered by polymerase chain reaction (PCR). The 5′ oligonucleotidehas the following structure: 5′-ACG CAT ATG GAG CGA GCT GAG AGC TCGAGT-3′ (SEQ ID NO: 1) that includes the NdeI restriction site CAT ATG.The 3′-oligonucleotide has the following structure: 5′-GAA TTC TTA TCACGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC AGT TCC AAC CTTCGC CGA TGC-3′ (SEQ ID NO: 2) that includes an EcoRI site GAA TTC andthe 14 amino acid epitope for the V5 antibody. The oligonucleotides wereused for PCR under standard conditions that included 25 cycles of PCR(95° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25cycles followed by 3 minutes at 72° C.). The PCR fragment was purifiedby gel electrophoresis and cloned into pTA2.1 (Invitrogen) by standardcloning methods (Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Edition, 2001), creating the plasmid pTA2.1-hPMS134.pTA2.1-hPMS134 was digested with the restriction enzyme EcoRI to releasethe insert which was cloned into EcoRI restriction site of pPIC3.5K(Invitrogen). The following strategy, similar to that described above toclone human PMS134, was used to construct an expression vector for thehuman related gene PMSR2. First, the hPMSR2 fragment was amplified byPCR to introduce two restriction sites, an NdeI restriction site at the5′-end and an Eco RI site at the 3′-end of the fragment. The5′-oligonucleotide that was used for PCR has the following structure:5′-ACG CAT ATG TGT CCT TGG CGG CCT AGA-3′ (SEQ ID NO: 3) that includesthe NdeI restriction site CAT ATG. The 3′-oligonucleotide used for PCRhas the following structure: 5′-GAA TTC TTA TTA CGT AGA ATC GAG ACC GAGGAG AGG GTT AGG GAT AGG CTT ACC CAT GTG TGA TGT TTC AGA GCT-3′ (SEQ IDNO: 4) that includes an EcoRI site GAA TTC and the V5 epitope to allowfor antibody detection. The plasmid that contained human PMSR3 inpBluescript SK (Nicolaides et al. Genomics 30 (2):195-206,1995) was usedas the PCR target with the hPMS2-specific oligonucleotides above.Following 25 cycles of PCR (95° C. for 1 minute, 55° C. for 1 minute,72° C. for 1.5 minutes for 25 cycles followed by 3 minutes at 72° C.).The PCR fragment was purified by gel electrophoresis and cloned intopTA2.1 (Invitrogen) by standard cloning methods (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, 2001), creatingthe plasmid pTA2.1-hR2. pTA2.1-hR2 was next digested with therestriction enzyme EcoRI to release the insert (there are two EcoRIrestriction sites in the multiple cloning site of pTA2.1 that flank theinsert) and the inserted into the yeast expression vector pPIC3.5K(Invitrogen).

Yeast cells were transformed with pPIC3.5K vector, pPIC3.5K-pms134, andpPIC3.5K-hR2 as follows. First, 5 ml of YPD (1% yeast extract, 2%bacto-peptone, 1% dextrose) medium was inoculated with a single colonyfrom a YPD plate (same as YPD liquid but add 2% difco-agar to plate) andincubated with shaking overnight at 30° C. The overnight culture wasused to inoculate 500 ml of YPD medium (200 ul of overnight culture) andthe culture incubated at 30° C. until the optical density at 600 nmreached 1.3 to 1.5. The cells were then spun down (4000×g for 10minutes), and then washed 2 times in sterile water (one volume eachtime), then the cells suspended in 20 ml of 1M sorbitol. Thesorbitol/cell suspension was spun down (4,000×g for 10 minutes) andsuspended in 1 ml of 1M sorbitol. 80 ul of the cell suspension was mixedwith 5 to 10 ug of linearized plasmid DNA and placed in a 0.2 cmcuvette, pulsed length 5 to 10 milliseconds at field strength of7,500V/cm. Next, the cells are diluted in 1 ml of 1M sorbitol andtransferred to a 15 ml tube and incubated at 30° C. for 1 to 2 hourswithout shaking. Next, the cells are spun out (4,000×G for 10 minutes)and suspended in 100 ul of sterile water, and 50 ul/plate spread ontothe appropriate selective medium plate. The plates are incubated for 2to 3 days at 30° C. and colonies patched out onto YPD plates for furthertesting.

Example 2 Generation of Hypermutable Yeast with Inducible DominantNegative Alleles of Mismatch Repair Genes

Yeast clones expressing human PMS2 homologue PMS-R2 or empty vector weregrown in BMG (100 mM potassium phosphate, pH 6.0, 1.34% YNB (yeastnitrogen base), 4×10⁻⁵% biotin, 1% glycerol) liquid culture for 24 hr at30° C. The next day, cultures were diluted 1:100 in MM medium (1.34%YNB, 4×10⁻⁵% biotin, 0.5% methanol) and incubated at 30° C. withshaking. Cells were removed for mutant selection at 24 and 48 hours postmethanol induction as described below (see EXAMPLE 3).

Example 3 Dominant Negative MMR Genes Can Produce New Genetic Variantsand Commercially Viable Output Traits in Yeast.

The ability to express MMR genes in yeast, as presented in example 2,demonstrate the ability to generate genetic alterations and newphenotypes in yeast expressing dominant negative MMR genes. In thisexample we teach the utility of this method to create eukaryotic strainswith commercially relevant output traits.

Generation of Uracil Dependent Yeast Strain

One example of utility is the generation of a yeast strain that ismutant for a particular metabolic product, such as an amino acid ornucleotide. Engineering such a yeast strain will allow for recombinantmanipulation of the yeast strain for the introduction of genes forscalable process of recombinant manufacturing. In order to demonstratethat MMR can be manipulated in yeast to generate mutants that lack theabilty to produce specific molecular building blocks, the followingexperiment was performed. Yeast cells that express a methanol induciblehuman PMS2 homologue, hPMS2-R2 (as described in example 1 above), weregrown in BMY medium overnight then diluted 1:100 and transferred to MMmedium, which results in activation of the AOX promoter and productionof the hPMS2-R2 MMR gene that is resident within the yeast cell. Controlcells were treated the same manner; these cells contain the pPIC3.5vector in yeast and lack an insert. Cells were induced for 24 and 48hours and then selected for uracil requiring mutations as follows. Thecells were plated to 5-FOA medium (Boeke, J. D., LaCroute, F., and Fink,G. R. Mol. Gen. Genet. 197:345-345,1984). The plates are made asfollows: (2×concentrate (filter sterilize): yeast nitrogen base 7 grams;5-fluoro-orotic acid 1 gram; uracil 50 milligrams; glucose 20 grams;water to 500 ml; Add to 500 ml 4% agar (autoclaved) and pour plates.Cells are plated on 5-FOA plates (0, 24 and 48 hour time points) andincubated at 30° C. for between 3 and 5 days. Data from a typicalexperiment is shown in Table 1. No uracil requiring clones were observedin the un-induced or induced culture in yeast cells that harbor the“empty” vector whereas those cells that harbor the MMR gene hPMS2-R2have clones that are capable of growth on the selection medium. Notethat the un-induced culture of hPMS2-R2 does not have any colonies thatare resistant to 5-FOA, demonstrating that the gene must be induced forthe novel phenotype to be generated. It has been demonstrated that themutagens (such as ethyl methyl sulfonate result in a low number of ura⁻mutants and that the spontaneous mutation rate for generating this classof mutants is low (Boeke, J. D., LaCroute, F. and Fink, G. R. Mol. Gen.Genet. 197:345-346,1984).

TABLE 1 Generation of uracil requiring mutant yeast cells. # Representsat 24 hour methanol induction and @ a 48 hour induction. For comparisona wild type yeast cell treated/un-treated is shown (Galli, A. andSchiestl, R.H. Mutat. Res. 429(1):13-26,1999). Frequency (ura⁻ StrainSeeded ura⁻ URA⁺ cells) Wt 100,000  0 ˜100,000 0 Empty 100,000  0˜100,000 0 pMOR^(ye-1#) 100,000 14 ˜100,000 1/7,142 pMOR^(ye2@) 100,000123  ˜100,000 1/813 Wt 100,000 1-0.1 100,000 1/10⁵⁻⁶* Mutagen 100,000 10100,000 1/10,000

Generation of Heat-resistant Producer Strains

One example of commercial utility is the generation of heat-resistantrecombinant protein producer strains. In the scalable process ofrecombinant manufacturing, large-scale fermentation of both prokaryotesand eukaryotes results in the generation of excessive heat within theculture. This heat must be dissipated by physical means such as usingcooling jackets that surround the culture while it is actively growingand producing product. Production of a yeast strain that can resist hightemperature growth effectively would be advantageous for large-scalerecombinant manufacturing processes. To this end, the yeast strain asdescribed in EXAMPLE 2 can be grown in the presence of methanol toinduce the dominant negative MMR gene and the cells grown for varioustimes (e.g. 12, 24, 36 and 48 hours) then put on plates and incubated atelevated temperatures to select for mutants that resist high temperaturegrowth (e.g. 37° C. or 42° C.). These strains would be useful forfermentation development and scale-up of processes and should result ina decrease in manufacturing costs due to the need to cool thefermentation less often.

Generation of High Recombinant Protein Producer Strains and Strains withLess Endogenous Protease Activity

Yeast is a valuable recombinant-manufacturing organism since it is asingle celled organism that is inexpensive to grow and easily lendsitself to fermentation at scale. Further more, many eukaryotic proteinsthat are incapable of folding effectively when expressed in Escherichiacoli systems fold with the proper conformation in yeast and arestructurally identical to their mammalian counterparts. There areseveral inherent limitations of many proteins that are expressed inyeast including over and/or inappropriate glycosylation of therecombinant protein, proteolysis by endogenous yeast enzymes andinsufficient secretion of recombinant protein from the inside of theyeast cell to the medium (which facilitates purification). To generateyeast cells that with this ability to over-secrete proteins, or withless endogenous protease activity and or less hyper-glycosylationactivity yeast cells as described in example 1 can be grown withmethanol for 12, 24, 36 and 48 hours and yeast cells selected for theability to over-secrete the protein or interest, under-glycosylate it ora cell with attenuated of no protease activity. Such a strain will beuseful for recombinant manufacturing or other commercial purposes andcan be combined with the heat resistant strain outlined above. Forexample, a mutant yeast cell that is resistant to high temperaturegrowth and can secrete large amounts of protein into the medium wouldresult.

Similar results were observed with other dominant negative mutants suchas the PMSR2, PMSR3, and the human MLH1 proteins.

Example 4 Mutations Generated in the Host Genome of Yeast by DefectiveMMR are Genetically Stable

As described in example 3 manipulation of the MMR pathway in yeastresults in alterations within the host genome and the ability to selectfor a novel output traits, for example the ability of a yeast cell torequire a specific nutrient. It is important that the mutationsintroduced by the MMR pathway is genetically stable and passed todaughter cells reproducibly once the wild type MMR pathway isre-established. To determine the genetic stability of mutationsintroduced into the yeast genome the following experiment was performed.Five independent colonies from pPIC3.5K-hPMS2-R2 that are ura⁻, fivewild type control cells (URA⁺) and five pPIC3.5K transformed cells(“empty vector”) were grown overnight from an isolated colony in 5 ml ofYPD (1% yeast extract, 2% bacto-peptone and 1% dextrose) at 30° C. withshaking. The YPD medium contains all the nutrients necessary for yeastto grow, including uracil. Next, 1 μL of the overnight culture, whichwas at an optical density (OD) as measured at 600 nM of>3.0, was dilutedto an OD₆₀₀ of 0.01 in YPD and the culture incubated with shaking at 30°C. for an additional 24 hours. This process was repeated 3 more timesfor a total of 5 overnight incubations. This is the equivalent ofgreater than 100 generations of doublings (from the initial colony onthe plate to the end of the last overnight incubation. Cells (fiveindependent colonies that are ura⁻ and five that were wild type werethen plated onto YPD plates at a cell density of 300 to 1,000cells/plate and incubated for two days at 30° C. The cells from theseplates were replica plated to the following plates and scored for growthfollowing three days incubation at 30° C.; Synthetic Complete (SC)SC-ura (1.34% yeast nitrogen base and ammonium sulfate; 4×10⁻⁵% biotin;supplemented with all amino acids, NO supplemental uracil; 2% dextroseand 2% agar); SC+URA (same as SC−ura but supplement plate with 50 mguracil/liter medium), and YPD plates. They were replica plated in thefollowing order—SC−ura, SC complete, YPD. If the novel output trait thatis resident within the yeast genome that was generated by expression ofthe mutant MMR (in this example the human homologue of PMS2, hPMS2-R2)is unstable, the uracil dependent cells should “revert” back a uracilindependent phenotype. If the phenotype is stable, growth of the mutantcells under non-selective conditions should result in yeast cells thatmaintain their viability dependence on exogenous supplementation withuracil. As can be seen in the data presented in Table 2, the uracildependent phenotype is stable when the yeast cells are grown undernon-selective conditions, demonstrating that the MMR-generated phenotypederived from mutation in one of the uracil biosynthetic pathway genes isstable genetically.

Strain Seeded −ura +URA YPD Wt 650 650 650 650 Empty 560 560 560 560pMOR^(ye-1#) 730  0 730 730

These data demonstrate the utility of employing an inducible expressionsystem and a dominant negative MMR gene in a eukaryotic system togenerate genetically altered strains. The strain developed in thisexample, a yeast strain that now requires addition of uracil for growth,is potentially useful as a strain for recombinant manufacturing; byconstructing an expression vector that harbors the wild type URA3 geneon either an integration plasmid or an extra-chromosomal vector it isnow possible to transform and create novel cells expressing the aprotein of interest. It is also possible to modify other resident genesin yeast cells and select for mutations in genes that that give otheruseful phenotypes, such as the ability to carry out a novelbio-transformation. Furthermore, it is possible to express a geneextra-chromosomally in a yeast cell that has altered MMR activity asdescribed above and select for mutations in the extra-chromosomal gene.Therefore, in a similar manner to that described above the mutant yeastcell can be put under specific selective pressure and a novel proteinwith commercially important biochemical attributes selected. Theseexamples are meant only as illustrations and are not meant to limit thescope of the present invention. Finally, as described above once amutation has been introduced into the gene of interest the MMR activityis attenuated of completely abolished. The result is a yeast cell thatharbors a stable mutation in the target gene(s) of interest.

Example 5 Enhanced Generation of MMR-Defective Yeast and ChemicalMutagens for the Generation of New Output Traits

It has been previously documented that MMR deficiency yields toincreased mutation frequency and increased resistance to toxic effectsof chemical mutagens (CM) and their respective analogues such as but notlimited to those as: ethidium bromide, EMS, MNNG, MNU, Tamoxifen,8-Hydroxyguanine, as well as others listed but not limited to inpublications by: Kibromov-Borisov, N. N., et al. Mutat. Res. 430:55-74,1999; Ohe, T., et al. (Mutat. Res. 429:189-199, 1999; Hour, T. C. et al.Food Chem. Toxicol. 37:569-579, 1999; Hrelia, P., et al. Chem. Biol.Interact. 118:99-111, 1999; Garganta, F., et al. Environ. Mol. Mutagen.33:75-85, 1999; Ukawa-Ishikawa S., et al. Mutat. Res. 412:99-107, 1998;the web site havina the URL address: www host server, ehs.utah.edudomain name, ohh directory, mutaaens subdirectory; Marcelino L A, AndreP C, Khrapko K, Coller H A, Griffith J, Thilly W G. Chemically inducedmutations in mitochondrial DNA of human cells: mutational spectrum ofN-methyl-N′-nitro-N-nitrosoguanidine. Cancer Res Jul. 1,1998;58(13):2857-62; Koi M, Umar A, Chauhan D P, Cherian S P, CarethersJ M, Kunkel T A, Boland C R. Human chromosome 3 corrects mismatch repairdeficiency and microsatellite instability and reducesN-methyl-N′-nitro-N-nitrosoguanidine tolerance in colon tumor cells withhomozygous hMLH1 mutation. Can Res 1994 54:4308-4312,1994. Mismatchrepair provokes chromosome aberrations in hamster cells treated withmethylating agents or 6-thioguanine, but not with ethylating agents. Todemonstrate the ability of CMs to increase the mutation frequency in MMRdefective yeast cells, we would predict that exposure of yeast cells toCMs in the presence or absence of methanol (which induces the expressionof the resident human homologue to PMS2, hPMS2-R2) will result in anaugmentation of mutations within the yeast cell.

Yeast cells that express hPMS2-R2 (induced or un-induced) and emptyvector control cells are grown as described in examples 2 and 3) and for24 hours and diluted into MM medium as described above. Next, the cellsin MM are incubated either with or without increasing amounts of ethylmethane sulfonate (EMS) from 0, 1, 10, 50, 100, and 200 μM. 10 μLaliquots of culture (diluted in 300 μMM) and incubated for 30 minutes,60 minutes, and 120 minutes followed by plating cells onto 5-FOA platesas described in example 3 above. Mutants are selected and scored asabove. We would predict that there will be an increase in the frequencyof ura⁻ mutants in the PMS2-R2 cultures that are induced with methanolas compared to the uninduced parental or wild type strain. In a furtherextension of this example, human PMS2-R2 harboring cells will be inducedfor 24 and 48 hours then mutagenized with EMS. This will allow the MMRgene to be filly active and expressed at high levels, thereby resultingin an increase in the number of ura⁻ mutants obtained. We would predictthat there will be no change in the number of ura⁻ mutants obtained inthe un-induced parental control or the wild type “empty vector” cells.

This example demonstrates the use of employing a regulated dominantnegative MMR system plus chemical mutagens to produce enhanced numbersof genetically altered yeast strains that can be selected for new outputtraits. This method is useful for generating such organisms forcommercial applications such as but not limited to recombinantmanufacturing, biotransformation, and altered biochemicals with enhancedactivities. It is also useful to obtain alterations of protein activityfrom ectopically expressed proteins harbored on extra-chromosomalexpression vectors similar to those described in example 4 above.

Example 6 Alternative Methods to Inhibition of Yeast MMR Activity

The inhibition of MMR activity in a host organism can be achieved byintroducing a dominant negative allele as shown in the examples above.This application also teaches us the use of using regulated systems tocontrol MMR in yeast to generate genetic diversity and output traits forcommercial applications. Additional methods to regulate the suppressionof MMR activity of a host are by using genetic recombination to knockout alleles of a MMR gene within the cell of interest. This can beaccomplished by use of homologous recombination that disrupts theendogenous MMR gene; 2) blocking MMR protein dimerization with othersubunits (which is required for activity) by the introduction ofpolypeptides or antibodies into the host via transfection methodsroutinely used by those skilled in the art (e.g. electroporation); or 3)decreasing the expression of a MMR gene using anti-senseoligonucleotides.

MMR gene knockouts. We intend to generate disrupted targeting vectors ofa particular MMR gene and introduce it into the genome of yeast usingmethods standard in the art. Yeast exhibiting hypennutability will beuseful to produce genetically diverse offspring for commercialapplications. Yeast will be confirmed to have lost the expression of theMMR gene using standard northern and biochemical techniques (asdescribed in reference 31). MMR gene loci can be knocked out, strainsselected for new output traits and MMR restored by introducing a wildtype MMR gene to complement the KO locus. Other strategies include usingKO vectors that can target a MMR gene locus, select for host outputtraits and then have the KO vector “spliced” from the genome afterstrain generation.

Blocking peptides. MMR subunits (MutS and MutL proteins) interact toform active MMR complexes. Peptides are able to specifically inhibit thebinding of two proteins by competitive inhibition. Introduction intocells of peptides or antibodies to conserved domains of a particular MMRgene to disrupt activity is straightforward to those skilled in the art.Yeast will be verified for loss of expression of the MMR activity bystandard northern and/or biochemical techniques (as described inNicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998.A naturally occurring hPMS2 mutation can confer a dominant negativemutator phenotype. Mol Cell Biol 18:1635-1641). Yeast exhibitinghypermutability will be useful to produce genetically diverse sibs forcommercial applications.

Discussion

The results described above will lead to several conclusions. First,expression of dominant negative MMR proteins results in an increase inmicrosatellite instability and hypermutability in yeast. Thehypermutability of the yeast cell is due to the inhibition of theresident, endogenous MMR biochemical activity in these hosts. Thismethod provides a claim for use of MMR genes and their encoded productsfor the creation of hypermutable yeast to produce new output traits forcommercial applications.

Examples of MMR Genes and Encoded Polypeptides

Yeast MLH1 cDNA (accession number U07187) (SEQ ID NO: 5)

Yeast MLH1 cDNA (accession number U07187) (SEQ ID NO:5)    1 aaataggaatgtgatacctt ctattgcatg caaagatagt gtaggaggcg ctgctattgc   61 caaagacttttgagaccgct tgctgtttca ttatagttga ggagttctcg aagacgagaa  121 attagcagttttcggtgttt agtaatcgcg ctagcatgct aggacaattt aactgcaaaa  181 ttttgatacgatagtgatag taaatggaag gtaaaaataa catagaccta tcaataagca  241 atgtctctcagaataaaagc acttgatgca tcagtggtta acaaaattgc tgcaggtgag  301 atcataatatcccccgtaaa tgctctcaaa gaaatgatgg agaattccat cgatgcgaat  361 gctacaatgattgatattct agtcaaggaa ggaggaatta aggtacttca aataacagat  421 aacggatctggaattaataa agcagacctg ccaatcttat gtgagcgatt cacgacgtcc  481 aaattacaaaaattcgaaga tttgagtcag attcaaacgt atggattccg aggagaagct  541 ttagccagtatctcacatgt ggcaagagtc acagtaacga caaaagttaa agaagacaga  601 tgtgcatggagagtttcata tgcagaaggt aagatgttgg aaagccccaa acctgttgct  661 ggaaaagacggtaccacgat cctagttgaa gacctttttt tcaatattcc ttctagatta  721 agggccttgaggtcccataa tgatgaatac tctaaaatat tagatgttgt cgggcgatac  781 gccattcattccaaggacat tggcttttct tgtaaaaagt tcggagactc taattattct  841 ttatcagttaaaccttcata tacagtccag gataggatta ggactgtgtt caataaatct  901 gtggcttcgaatttaattac ttttcatatc agcaaagtag aagatttaaa cctggaaagc  961 gttgatggaaaggtgtgtaa tttgaatttc atatccaaaa agtccatttc attaattttt 1021 ttcattaataatagactagt gacatgtgat cttctaagaa gagctttgaa cagcgtttac 1081 tccaattatctgccaaaggg cttcagacct tttatttatt tgggaattgt tatagatcCg 1141 gcggctgttgatgttaacgt tcacccgaca aagagagagg ttcgtttcct gagccaagat 1201 gagatcatagagaaaatcgc caatcaattg cacgccgaat tatctgccat tgatacttca 1261 cgtactttcaaggcttcttc aatttcaaca aacaagccag agtcattgat accatttaat 1321 gacaccatagaaagtgatag gaataggaag agtctccgac aagcccaagt ggtagagaat 1381 tcatatacgacagccaatag tcaactaagg aaagcgaaaa gacaagagaa taaactagtc 1441 agaatagatgcttcacaagc taaaattacg tcatttttat cctcaagtca acagttcaac 1501 tttgaaggatcgtctacaaa gcgacaactg agtgaaccCa aggtaacaaa tgtaagccaC 1561 tcccaagaggcagaaaagct gacactaaat gaaagcgaac aaccgcgtga tgccaataca 1621 atcaatgataatgacttgaa ggatcaacct aagaagaaac aaaagttggg ggattataaa 1681 gttccaagcattgccgatga cgaaaagaat gcactcccga tttcaaaaga cgggtatatt 1741 agagtacctaaggagcgagt taatgttaat cttacgagta tcaagaaatt gcgtgaaaaa 1801 gtagatgattcgatacatcg agaactaaca gacatttttg caaatttgaa ttacgttggg 1861 gttgtagatgaggaaagaag attagccgct attcagcatg acttaaagct ttttttaata 1921 gattacggatctgtgtgcta tgagctattc tatcagattg gtttgacaga cttcgcaaac 1981 tttggtaagataaacctaca gagtacaaat gtgtcagatg atatagtttt gtataatctc 2041 ctatcagaatttgacgagtt aaatgacgat gcttccaaag aaaaaataat tagtaaaata 2101 tgggacatgagcagtatgct aaatgagtac tattocatag aattggtgaa tgatggtcta 2161 gataatgacttaaagtctgt gaagctaaaa tctctaccac tacttttaaa aggctacatt 2221 ccatctctggtcaagttacc attttttata tatcgcctgg gtaaagaagt tgattgggag 2281 gatgaacaagagtgtctaga tggtatttta agagagattg cattactcta tatacctgat 2341 atggttccgaaagtcgatac actcgatgca tcgttgtcag aagacgaaaa agcccagttt 2401 ataaatagaaaggaacacat atcctcatta ctagaacacg ttctcttccc ttgtatcaaa 2461 cgaaggttcctggcccctag acacattctc aaggatgtcg tggaaatagc caaccttcca 2521 gatctatacaaagtttttga gaggtgttaa ctttaaaacg ttttggctgt aataccaaag 2581 tttttgtttatttcctgagt gtgattgtgt ttcatttgaa agtgtatgcc ctttccttta 2641 acgattcatccgcgagattt caaaggatat gaaatatggt tgcagttagg aaagtatgtc 2701 agaaatgtatattcggattg aaactcttct aatagttctg aagtcacttg gttccgtatt 2761 gttttcgtcctcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gtaccaaaga 2821 cccgagtccttttatgagag aaaacatttc atcatttttc aactcaatta tcttaatatc 2881 attttgtagtattttgaaaa caggatggta aaacgaatca cctgaatcta gaagctgtac 2941 cttgtcccataaaagtttta atttactgag cctttcggtc aagtaaacta gtttatctag 3001 ttttgaaccgaatattgtgg gcagatttgc agtaagttca gttagatcta ctaaaagttg 3061 tttgacagcagccgattcca caaaaatttg gtaaaaggag atgaaagaga cctcgcg 3121 aatggtttgcatcaccatcg gatgtctgtt gaaaaactca ctttttgcat ggaagtt 3181 aacaataagactaatgatta ccttagaata atgtataa Yeast MLH1 protein (accession numberU07187) (SEQ ID NO:15) MSLRIKALDASVVNKIAAGEIIISPVNALKEMMENSIDANATMIDILVKEGGIKVLQITDNGSGINKADLPILCERFTTSKLQKFEDLSQIQTYGFRGEALASISHVARVTVTTKVKEDRCAWRVSYAEGKMLESPKPVAGKDGTTILVEDLFFNIPSRLRALRSHNDEYSKILDVVGRYAIHSKDIGFSCKKFGDSNYSLSVKPSYTVQDRIRTVFNKSVASNLITFHISKVEDLNLESVDGKVCNLNFISKKSISLIFFINNRLVTCDLLRRALNSVYSNYLPKGFRPFIYLGIVIDPAAVDVNVHPTKREVRFLSQDEIIEKIANQLHAELSAIDTSRTFKASSISTNKPESLIPFNDTIESDRNRKSLRQAQVVENSYTTANSQLRKAKRQENKLVRIDASQAKITSFLSSSQQFNFEGSSTKRQLSEPKVTNVSHSQEAEKLTLNESEQFRDANTINDNDLKDQPKKKQKLGDYKVFSIADDEKNALPISKDGYIRVPKERVNVNLTSIKKLREKVDDSIHRELTDIFANLNYVGVVDEERRLAAIQHDLKLFLIDYGSVCYELFYQIGLTDFANFGKINLQSTNVSDDIVLYNLLSEFDELNDDASKEKIISKIWDMSSMLNEYYSIELVNDGLDNDLKSVKLKSLPLLLKGYIPSLVKLPFFIYRLGKEVDWEDEQECUDGIURKIALLYIPDMVFKVDTLDASLSEDEKAQFINRKEHISSLLEHVLFPCIKRRFLAPRHILKDVVEIANLPDLYKVFERC Mouse PMS2 protein (SEQ ID NO:16)MEQTEGVSTE CAKAIKPIDG KSVHQICSGQ VILSLSTAVK ELIENSVDAG ATTIDLRLKD 60YGVDLIEVSD NGCGVEEENF ECLALKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120TISTCHGSAS VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE 180YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SGMKENIGSV FGQKQLQSLI 240PFVQLPFSDA VOEZYGUSTS GRHKTFSTFR ASFHSARTAP GGVQQTGSFS SSIRGPVTQQ 300RSLSLSMRFY HMYNRNQYPF VVLNVSVDSE CVDINVTPDK RQILLQEEKL LLAVLKTSLI 360GMFDSDANKL NVNQQPLLDV EGNLVKLHTA ELEKPVPGKQ DNSPSLKSTA DEKRVASISR 420LREAFSLHPT KEIKSRGFET AELTRSFFSE KRGVLSSYPS DVISYRGLRG SQDKLVSFTD 480SPGDCMDREK IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL 540DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA 600EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC PGENQAAEDE 660LRKEISKSMF AEMEILGQFN LGFIVTKLKE DLFLVDQHAA DEKYNFEMLQ QBTVLQAQRL 720ITFQTLNLTA VNEAVLIENL EIFRKNGFDF VIDEDAPVTE RAKLISLPTS KNWTFGPQDI 780DELIFMLSDS PGVMCRPSRV RQMFASRACR KSVMIGTALN ASEMKKLITH MGEMDHPWNC 840PHGRFTMRHV ANUDVISQN 859 Mouse PMS2 cDNA (SEQ ID NO:6) gaattccggtgaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcgtcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120 gtcttttcccgagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg 180 catccatggagcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240 atgggaagtcagtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg 300 tgaaggagttgatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta 360 aagactatggggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaaggtctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480 cgcaggttgaaactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg 540 atgtcactatatctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600 ataatgggaaaatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg 660 tgcagcacttattttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa 720 aggagtattccaaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctgcactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840 gcacgtctggcatgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttttgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacgccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020 cgccgggaggagtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtctctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgtccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200 ataaaaggcaaattctacta caagaagaga agctattgct ggccgtttta aagacctcct 1260 tgataggaatgtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag 1320 atgttgaaggtaacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa 1380 agcaagataactctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct 1440 ccaggctgagagaggccttt tctcttcatc ctactaaaga gatcaagtCt aggggtccag 1500 agactgctgaactgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560 cttcagacgtcatctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccctggtgactgt atggacagag agaaaataga aaaagactca gggctcagca 1680 gcacctcagctggctctgag gaagagttca gcaccccaga agtggccagt agctttagca 1740 gtgactataacgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg 1800 acctggactgccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagctctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920 aggaaagaccctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggtcgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040 tgagttctctagctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg 2100 aactgagttacagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcagaaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctgggatttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280 ctgcggatgagaagtacaac tttgagatgc tgcagcagca cacggtgcto caggcgcaga 2340 ggctcatcacaccccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaatattcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca 2460 ctgaaagggctaaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatgaactgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagacagatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640 tcaatgcgagcgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga 2700 actgcccccacggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga 2760 actgacacaccccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg 2820 ttttaagtaatctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc 2880 catttaaaagcagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtgggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000 agactcaattcaaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056 human PMS2protein (SEQ ID NO:7) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSVDVKLENYGFD KIEVRDNGEG 60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSICCIAEVLITT RTAADNFSTQ 120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYSTAKKCKDEIK KIQDLLMSFG 180 ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMNNMESFQYHSE ESQIYLSGFL 240 PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHYNLKCLKESTR LYPVFFLKID 300 VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPLPSTNSYENNK TDVSAADIVL 360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESGKNTDDCLNHQ ISIGDFGYGH 420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFISSVKHTQSENG NKDHIDESGE 480 NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVPEKSLPCKVSN NNYPIPEQMN 540 LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASALFVQDHRPQFL IENPKTSLED 600 ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIEQESQMSLKDG RKKIKPTSAW 660 NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIPFSMKNLKINF KKQNKVDLEE 720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKRLLENHKLPAE PLEKPIMLTE 780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKIKLIPGVSITE NYLEIEGMAN 840 CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGEAVRLSRQLPM YLSKEDIQDI 900 IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT 932 HumanPMS2 CDNA (SEQ ID NO:17) cgaggcggat cgggtgttgc atccatggag cgagctgagagctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca gtccatcagatttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaacagtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggaotatgga gtggatcttattgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctgaaacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggctttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgccacgcatcggc gaaggttgga 420 actcgactga tgtttgatca caatgggaaa attatccagaaaacccccta cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta ttttccacactacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtccaggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc accaatcagcttggacaagg aaaacgacag 660 cctgtggtat gcacaggtgg aagccccagC ataaaggaaaatatcggctc tgtgtttggg 720 cagaagcagt tgcaaagcct cattcctttt gttcagctgccccctagtga ctccgtgtgt 780 gaagagtacg gtttgagctg ttcggatgct ctgcataatcttttttacat ctcaggtttc 840 atttcacaat gcacgcatgg agttggaagg agttcaacagacagacagtt tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcgtgaatgaggt ctaccacatg 960 tataatcgac accagtatcc atttgttgtt cttaacatttctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga taaaaggcaa attttgctacaagaggaaaa gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtgatgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt aacttaataaaaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcattaaggactgg agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctcttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca agaaggagccctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaaggcgtcctgag acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacggacagagogga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat tctgaggggttcagcatccc agacacgggc 1560 agtcactgca gcagcgagta tgcggccagc tccccaggggacaggggctc gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctttttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt cgagttttgcctcagccaac taatctcgca 1740 accccaaaca caaagcgttt taaaaaagaa gaaattctttccagttctga catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttgatgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg agttctttagctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag tgaaggggaa cagaattacaggaagtttag ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaaaagagataag taaaacgatg 2040 tttgcagaaa tggaaatcat tggtcagttt aacctgggatttataataac caaactgaat 2100 gaggatatct tcatagtgga ccagcatgcc acggacgagaagtataactt cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcacctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat ctggaaatatttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc tccagtcact gaaagggctaaactgatttc cttgccaact 2340 agtaaaaact ggaccttcgg accccaggac gtcgatgaactgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga gtcaagcagatgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcgagatgaagaa actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatggaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac tgaccgtagtcactgtatgg aataattggt 2640 tttatcgcag atttttatgt tttgaaagac agagtcttcactaacctttt ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccatttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 human PMS1 protein (SEQ IDNO:18) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ 120YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG 180ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL 240PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID 300VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL 360SKTAETDVLF NKVESSGKNY SUVDTSVIPF QNDMHNDESG KNTDDCLNHQ ISIGDFGYGH 420CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS SVKHTQSENG NKDHIDESGE 480NEEHAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP EKSLPCKVSN NNYPIPEQMN 540LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED 600ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKHAIE QESQMSLKDG RKKIKPTSAW 660NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSNKNLKINF KKQNKVDLEE 720KDEPCLIHNL RFPDAWLMTS KTEVNLLNPY RVEEALLFKR LLENHKLPAE PLEKPIMLTE 780SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI KLIPGVSITE NYLEIEGMAN 840CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE AVRLSRQLPM YLSKEDIQDI 900IYENKHQFGN EIKECVHGRP FFHHLTYLPE TT 932 Human PMS1 cDNA (SEQ ID NO:8)ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa 120gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg 180atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg 240tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact 300acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac 480cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg 540taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag 600atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca 660aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc 720tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa 840caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa 900agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg 960ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata 1020aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg 1200aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata 1260tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg 1320gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga 1380atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag acggaatata 1440gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt 1560ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac 1620ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc 1680caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag 1740ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac 1800ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg 1920aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc 1980aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga 2040taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta 2100atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg 2280atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag 2340aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa 2400agccaattat gttaacagag agtcttttta atggatctca ttatttagac gttttatata 2460aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta 2520cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc 2640ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga 2700taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa 2760aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag 2820agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat 2880taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca 3000ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat 3060aac 3063 human MSH2 protein (SEQ ID NO:19) MAVQPKETLQ LESAAEVGFVRFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT 60 QGVIKYMGPA GAKNLQSVVLSKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA 120 YKASPGNLSQ FEDILFGNNDMSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ RKLGLCEFPD 180 NDQFSNLEAL LIQIGPKECVLPGGETAGDM GKLRQIIQRG GILITERKKA DFSTKDIYQD 240 LNRLLKGKKG EQMNSAVLPEMENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM 300 KLDIAAVRAL NLFQGSVEDTTGSQSLAALL NKCKTPQGQR LVNQWIKQPL MDKNRIEERL 360 NLVEAFVEDA ELRQTLQEDLLRRFPDLNRL AKKFQRQAAN LQDCYRLYQG INQLPNVIQA 420 LEKHEGKHQK LLLAVFVTPLTDLRSDFSKF QEMIETTLDM DQVENHEFLV KPSFDPNLSE 480 LREIMNDLEK KMQSTLISAARDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS 540 TVDIQKNGVK FTNSKLTSLNEEYTKNKTEY EEAQDAIVKE IVNISSGYVE PNQTLNDVLA 600 QLDAVVSFAH VSNGAPVPYVRPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD 660 KQMFHIITGP NMGGKSTYIRQTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK 720 GVSTFMAEML ETASILRSATKDSLIIIDEL GRGTSTYDGF GLAWAISEYI ATKIGAFCMF 780 ATHFHELTAL ANQIPTVNNLHVTALTTEET LTMLYQVKKG VCDQSFGIHV AELANFPKHV 840 LECAKQKALE LEEFQYIGESQGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS 900 EENITIKLKQ LKAEVIAKNNSFVNEIISRI KVTT 934 Human MSH2 cDNA (SEQ ID NO:9) ggcgggaaac agcttagtgggtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacat ggcggtgcagccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120 gcttcgtgcg cttctttcagggcatgccgg agaagccgac caccacagtg cgccttttcg 180 accggggcga cttctatacggcgcacggcg aggacgcgct gctggccgcc cgggaggtgt 240 Lcaagaccca gggggtgatcaagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag taaaatgaattttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360 atagagttga agtttataagaatagagctg gaaataaggc atccaaggag aatgattggt 420 atttggcata taaggcttctcctggcaatc tctctcagtt tgaagacatt ctctttggta 480 acaatgatat gtcagcttccattggtgttg tgggtgttaa aatgtccgca gttgatggcc 540 agagacaggt tggagttgggtatgtggatt coatacagag gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttctccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt acccggaggagagactgctg gagacatggg gaaactgaga cagataattc 720 aaagaggagg aattctgatcacagaaagaa aaaaagctga cttttccaca aaagacattt 780 atcaggacct caaccggttgttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840 tgccagaaat ggagaatcaggttgcagttt catcactgtc tgcggtaatc aagtttttag 900 aactcttatc agatgattccaactttggac agtttgaact gactactttt gacttcagcc 960 agtatatgaa attggatattgcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac tggctctcagtctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccagtggattaagc agcctctcat ggataagaac agaatagagg 1140 agagattgaa tttagtggaagcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttact tcgtcgattcccagatctta accgacttgc caagaagttt caaagacaag 1260 cagcaaactt acaagattgttaccgactct atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacatgaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac tgatcttcgttctgacttct ccaagtttca ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaaaaccatgaat tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt aagagaaataatgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccag agatcttggcttggaccctg gcaaacagat taaactggat tccagtgcac 1620 agtttggata ttactttcgtgtaacctgta aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatccagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga agagtataccaaaaataaaa cagaatatga agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatttcttcaggct atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca gctagatgctgttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacg accagccattttggagaaag gacaaggaag aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagttcaagatgaaa ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttccacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca aactggggtgatagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccattgtggactgca tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacgttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa agattcattaataatcatag atgaattggg aagaggaact tctacctacg 2340 atggatttgg gttagcatgggctatatcag aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccattttcatgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca tgtcacagcactcaccactg aagagacctt aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaaagttttggga ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgctaaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca aggatatgatatcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattattcaggagttcc tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatcacaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag ctttgtaaatgaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaatattgataag ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccatagtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta atattttactttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060 gctgtaactg aggactgtttgcaattgaca taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtttgtgg 3145 human MLH1 protein (SEQ ID NO:20) MSFVAGVIRR LDETVVNRIAAGEVIQRPAN AIKEMIENCL DAKSTSIQVI VKEGGLKLIQ 60 IQDNGTGIRK EDLDIVCERFTTSKLQSFED LASISTYGFR GEALASISHV AHVTITTKTA 120 DGKCAYRASY SDGKLKAPPKPCAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV 180 GRYSVHNAGI SFSVKKQGETVADVRTLPNA STVDNIRSIF GNAVSRELIE IGCEDKTLAF 240 KMNGYISNAN YSVKKCIFLLFINHRLVEST SLRKAIETVY AAYLPKNTHP FLYLSLEISP 300 QNVDVNVHPT KHEVHFLHEESILERVQQHI ESKLLGSNSS RMYFTQTLLP GLAGPSGEMV 360 KSTTSLTSSS TSGSSDKVYAHQMVRTDSRE QKLDAFLQPL SKPLSSQPQA IVTEDKTDIS 420 SGRARQQDEE MUlLPAPAEVAAKNQSLEGD TTKGTSEMSE KRGPTSSNPR KRHREDSDVE 480 MVEDDSRKEM TAACTPRRRIINLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL 540 AQHQTKLYLL NTTKLSEELFYQILIYDFAN FGVLRLSEPA FLFDLAMLAL DSPESGWTEE 600 DGPKEGLAEY IVEFLKKKAEMLADYFSLEI DEEGNLIGLP LLIDNYVPPL EGLPIFILRL 660 ATEVNWDEEK ECFESLSKECAMFYSIRKQY ISEESTLSGQ QSEVPGSIPN SWKWTVEHIV 720 YKALRSHILP PKHFTEDGNILQLANLPDLY KVFERC 756 Human MLH1 cDNA (SEQ ID NO:10) cttggctcttctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag 60 acagtggtgaaccgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa 120 gagatgattgagaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180 ggaggcctgaagttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240 gatattgtatgtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt 300 atttctacctatggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt 360 actattacaacgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga 420 aaactgaaagcccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag 480 gaccttttttacaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540 gggaaaattttggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca 600 gttaaaaaacaaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg 660 gacaatattcgctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt 720 gaggataaaaccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg 780 aagaagtgcatcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga 840 aaagccatagaaacagtgta tgcagcctat ttgcccaaaa acacacacco attcctgtac 900 ctcagtttagaaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960 gttcacttcctgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggctccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct 1080 ggcccctctggggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140 agtagtgataaggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt 1200 gatgcatttctgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260 gaggataagacagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320 ctcccagcccctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag 1380 gggacttcagaaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat 1440 cgggaagattctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500 tgtaccccccggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagggacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620 gtgaatcctcagtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680 aagcttagtgaagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt 1740 ctcaggttatcggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800 gagagtggctggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860 tttctgaagaagaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920 gggaacctgattggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980 cctatcttcattcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040 gaaagcctcagtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag 2100 gagtcgaccctctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160 tggactgtggaacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220 ttcacagaagatggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgttaaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaagtgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400 cacttaagacttatacttgc cttctgatag tattccttta tacacagtgg attgattata 2460 aataaatagatgtgtcttaa cata 2484 hPMS2-134 protein (SEQ ID NO:21) MKQLPAATVRLLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG 60 IKAVDAPVMAMKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ 120 YVLDGSGHILSQK 133 hPMS2-134 cDNA (SEQ ID NO:11) cgaggcggat cgggtgttgc atccatggagcgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtcagtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagttagtagaaaaca gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatggagtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggcttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaaacttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatttctacctgcc acgcatcggc gaaggttgga 420 acttga 426 hMSH6 (human cDNA)ACCESSION U28946 (SEQ ID NO:22)MSRQSTLYSFFPKSPALSDANKASARASREGGRAAAAPGASPSPGGDAAWSEAGPGPRPLARSASPPKAKNLNGGLRRSVAPAAPTSCDFSPGDLVWAKNEGYPWWPCLVYNHPFDGTFIREKGKSVRVHVQFFDDSPTRGWVSKRLLKPYTGSKSKEAQKGGHFYSAKPEILRAMQRADEALNKDKIKRLELAVCDEPSEPEEEEEMEVGTTYVTDKSEEDNEIESEEEVQPKTQGSRRSSRQIKKRRVISDSESDIGGSDVEFKPDTKEEGSSDEISSGVGDSESEGLNSPVKVARKRKRMVTGNGSLKEKSSRKETPSATKQATSISSETKNTLRAFSAPQNSESQAHVSGGGDDSSRPTVWYHETLEWLKEEKRRDEHRRRPDHPDFDASTLYVPEDFLNSCTPGMRKWWQIKSQNFDLVICYKVGKFYELYHMDALIGVSELGLVFNKGNWAHSGFPEIAFGRYSDSLVQKGYKVARVEQTETPEMMEARCRKMAHISKYDRVVRREICRIITKGTQTYSVLEGDPSENYSKYLLSLKEKEEDSSGETRAYGVCFVDTSLGKFFIGQFSDDRHCSRFRTLVAHYPPVQVLFEKGNLSKETKTILKSSLSCSLQEGLIPGSQFWDASKTLRTLLEEEYFREKLSDGIGVMLPQVLKGMTSESDSIGLTPGEKSELALSALGGCVFYLKKCLIDQELLSMANFEEYIPLDSDTVSTTRSGAIFTKAYQRMVLDAVTLNNLEIFLNGTNGSTEGTLLERVDTCHTPFGKRLLKQWLCAPLCNHYAINDRLDAIEDLMVVPDKISEVVELLKKLPDLERLLSKIHNVGSPLKSQNHPDSRAIMYEETTYSKKKIIDFLSALEGFKVMCKIIGIMEEVADGFKSKILKQVISLQTKNPEGRFPDLTVELNRWDTAFDHEKARKTGLITPKAGFDSDYDQALADIRENEQSLLEYLEKQRNRIGCRTIVYWGIGRNRYQLEIPENFTTRNLPEEYELKSTKKGCKRYWTKTIEKKLANLINAEERRDVSLKDCMERLFYNFDKNYKDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVILLPEDTPPFLELKGSRHPCITKTFFGDDFIPNDILIGCEEEEQENGKAYCVLVTGPNMGGKSTLMRQAGLLAVMAQMGCYVPAEVCRLTPIDRVFTRLGASDRIMSGESTFFVELSETASILMHATAHSLVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNVAVRLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGHRKAREFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL” hPMSR2 (human cDNA) ACCESSIONU38964 (SEQ ID NO:12)    1 ggcgctccta cctgcaagtg gctagtgcca agtgctgggccgccgctcct gccgtgcatg   61 ttggggagcc agtacatgca ggtgggctcc acacggagaggggcgcagac ccggtgacag  121 ggctttacct ggtacatcgg catggcgcaa ccaaagcaagagagggtggc gcgtgccaga  181 caccaacggt cggaaaccgc cagacaccaa cggtcggaaaccgccaagac accaacgctc  241 ggaaaccgcc agacaccaac gctcggaaac cgccagacaccaaggctcgg aatccacgcc  301 aggccacgac ggagggcgac tacctccctt ctgaccctgctgctggcgtt cggaaaaaac  361 gcagtccggt gtgctctgat tggtccaggc tctttgacgtcacggactcg acctttgaca  421 gagccactag gcgaaaagga gagacgggaa gtattttttccgccccgccc ggaaagggtg  481 gagcacaacg tcgaaagcag ccgttgggag cccaggaggcggggcgcctg tgggagccgt  541 ggagggaact ttcccagtcc ccgaggcgga tccggtgttgcatccttgga gcgagctgag  601 aactcgagta cagaacctgc taaggccatc aaacctattgatcggaagtc agtccatcag  661 atttgctctg ggccggtggt accgagtcta aggccgaatgcggtgaagga gttagtagaa  721 aacagtctgg atgctggtgc cactaatgtt gatctaaagcttaaggacta tggagtggat  781 ctcattgaag tttcaggcaa tggatgtggg gtagaagaagaaaacttcga aggctttact  841 ctgaaacatc acacatgtaa gattcaagag tttgccgacctaactcaggt ggaaactttt  901 ggctttcggg gggaagctct gagctcactt tgtgcactgagtgatgtcac catttctacc  961 tgccgtgtat cagcgaaggt tgggactcga ctggtgtttgatcactatgg gaaaatcatc 1021 cagaaaaccc cctacccccg ccccagaggg atgacagtcagcgtgaagca gttattttct 1081 acgctacctg tgcaccataa agaatttcaa aggaatattaagaagaaacg tgcctgcttc 1141 cccttcgcct tctgccgtga ttgtcagttt cctgaggcctccccagccat gcttcctgta 1201 cagcctgtag aactgactcc tagaagtacc ccaccccacccctgctcctt ggaggacaac 1261 gtgatcactg tattcagctc tgtcaagaat ggtccaggttcttctagatg atctgcacaa 1321 atggttcctc tcctccttcc tgatgtctgc cattagcattggaataaagt tcctgctgaa 1381 aatccaaaaa aaaaaaaaaa aaaaaaaa hPMSR2 (humanprotein) ACCESSION U38964 (SEQ ID NO:23)MAQPKQERVARZARHQRSETARHQRSETAKTPTLGNRQTPTLGNRQTPRLGIHARFRRRATTSLLTLLLAFGKNAVRCALIGPGSLTSRTRPLTEPLGEKERREVFFPPRPERVEHNVESSRWEPRRRGACGSRGGNFPSPRGGSGVASLERAENSSTEPAKAIKPIDRKSVHQICSGPVVPSLRPNAVKELVENSLDAGATNVDLKLKDYGVDLIEVSGNGCGVEEENFEGFTLKHHTCKIQEFADLTQVETFGFRGEALSSLCALSDVTISTCRVSAKVGTRLVFDHYGKIIQKTPYPRPRGMTVSVKQLFSTLPVHHKEFQRNIKKKRACFPFAFCRDCQFPEASPIAI4LPVQPVELTPRSTPPHPCSLEDNVITVFSSVKNGPGSSR HPMSR3 (humancDNA) ACCESSION U38979 (SEQ ID NO:13)    1 tttttagaaa ctgatgtttattttccatca accatttttc catgctgctt aagagaatat   61 gcaagaacag cttaagaccagtcagtggtt gctcctaccc attcagtggc ctgagcagtg  121 gggagctgca gaccagtcttccgtggcagg ctgagcgctc cagtcttcag tagggaattg  181 ctgaataggc acagagggcacctgtacacc ttcagaccag tctgcaacct caggctgagt  241 agcagtgaac tcaggagcgggagcagtcca ttcaccctga aattcctcct tggtcactgc  301 cttctcagca gcagcctgctcttctttttc aatctcttca ggatctctgt agaagtacag  361 atcaggcatg aoctcccatgggtgttcacg ggaaatggtg ccacgcatgc gcagaacttc  421 ccgagccagc atccaccacattaaacccac tgagtgagct cccttgttgt tgcatgggat  481 ggcaatgtcc acatagcgcagaggagaatc tgtgttacac agcgcaatgg taggtaggtt  541 aacataagat gcctccgtgagaggcgaagg ggcggcggga cccgggcctg gcccgtatgt  601 gtccttggcg gcctagactaggccgtcgct gtatggtgag ccccagggag gcggatctgg  661 gcccccagaa ggacacccgcctggatttgc cccgtagccc ggcccgggcc cctcgggagc  721 agaacagcct tggtgaggtggacaggaggg gacctcgcga gcagacgcgc gcgccagcga  781 cagcagcccc gccccggcctctcgggagcc ggggggcaga ggctgcggag ccccaggagg  841 gtctatcagc cacagtctctgcatgtttcc aagagcaaca ggaaatgaac acattgcagg  901 ggccagtgtc attcaaagatgtggctgtgg atttcaccca ggaggagtgg cggcaactgg  961 accctgatga gaagatagcatacggggatg tgatgttgga gaactacagc catctagttt 1021 ctgtggggta tgattatcaccaagccaaac atcatcatgg agtggaggtg aaggaagtgg 1081 agcagggaga ggagccgtggataatggaag gtgaatttcc atgtcaacat agtccagaac 1141 ctgctaaggc catcaaacctattgatcgga agtcagtcca tcagatttgc tctgggccag 1201 tggtactgag tctaagcactgcagtgaagg agttagtaga aaacagtctg gatgctggtg 1261 ccactaatat tgatctaaagcttaaggact atggagtgga tctcattgaa gtttcagaca 1321 atggatgtgg ggtagaagaagaaaactttg aaggcttaat ctctttcagc tctgaaacat 1381 cacacatgta agattcaagagtttgccgac ctaactgaag ttgaaacttt cggttttcag 1441 ggggaagctc tgagctcactgtgtgcactg agcgatgtca ccatttctac ctgccacgcg 1501 ttggtgaagg ttgggactcgactggtgttt gatcacgatg ggaaaatcat ocaggaaacc 1561 ccctaccccc accccagagggaccacagtc agcgtgaagc agttattttc tacgctacct 1621 gtgcgccata aggaatttcaaaggaatatt aagaagacgt gcctgcttcc ccttcgcctt 1681 ctgccgtgat tgtcagtttcctgaggcctc cccagccatg cttcctgtac agcctgcaga 1741 actgtgagtc aattaaacctcttttcttca taaattaaaa aaaaa hPMSR3 (human protein) ACCESSION U38979 (SEQID NO:24) MCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNSLGEVDRRGPREQTPAPATAAPPRPLGSRGAEAAEPQECLSATVSACFQEQQEMNTLQCPVSFKDVAVDFTQEEWRQLDPDEKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKEVEQGEEPWIMEGEFPCQHSPEPAKAIKPIDRKSVHQICSGPVVLSLSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM” hPMSL9 (human cDNA)ACCESSION NM_005395 (SEQ ID NO:14)   1 atgtgtcctt ggcggcctag actaggccgtcgctgtatgg tgagccccag ggaggcggat  61 ctgggccccc agaaggacac ccgcctggatttgccccgta gcccggcccg ggcccctcgg 121 gagcagaaca gccttggtga ggtggacaggaggggacctc gcgagcagac gcgcgcgcca 181 gcgacagcag ccccgccccg gcctctcgggagccgggggg cagaggctgc ggagccccag 241 gagggtctat cagccacagt ctctgcatgtttccaagagc aacaggaaat gaacacattg 301 caggggccag tgtcattcaa agatgtggctgtggatttca cccaggagga gtggcggcaa 361 ctggaccctg atgagaagat agcatacggggatgtgatgt tggagaacta cagccatcta 421 gtttctgtgg ggtatgatta tcaccaagccaaacatcatc atggagtgga ggtgaaggaa 481 gtggagcagg gagaggagoc gtggataatggaaggtgaat ttccatgtca acatagtcca 541 gaacctgcta aggccatcaa acctattgatcggaagtcag tccatcagat ttgctctggg 601 ccagtggtac tgagtctaag cactgcagtgaaggagttag tagaaaacag tctggatgct 661 ggtgccacta atattgatct aaagcttaaggactatggag tggatctcat tgaagtttca 721 gacaatggat gtggggtaga agaagaaaactttgaaggct taatctcttt cagctctgaa 781 acatcacaca tgtaa hPMSL9 (humanprotein) ACCESSION NN_005395 (SEQ ID NO:25)NCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNSLGEVDRRGPREQTRAPATAAPPRPLGSRGAEAAEPQEGLSATVSACFQEQQEMNTLQGPVSFKDVAVDFTQEEWRQLDPDEKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKEVEQGEEPWIMEGEFPCQHSPEPAKAIKPIDRKSVHQICSGPVVLSLSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM”

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 25 <210> SEQ ID NO 1 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 1acgcatatgg agcgagctga gagctcgagt          #                  #           30 <210> SEQ ID NO 2 <211> LENGTH: 75 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 2gaattcttat cacgtagaat cgagaccgag gagagggtta gggataggct ta#ccagttcc     60 aaccttcgcc gatgc               #                  #                   #    75 <210> SEQ ID NO 3 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 3acgcatatgt gtccttggcg gcctaga           #                  #             27 <210> SEQ ID NO 4 <211> LENGTH: 75 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 4gaattcttat tacgtagaat cgagaccgag gagagggtta gggataggct ta#cccatgtg     60 tgatgtttca gagct               #                  #                   #    75 <210> SEQ ID NO 5 <211> LENGTH: 3218<212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 5aaataggaat gtgatacctt ctattgcatg caaagatagt gtaggaggcg ct#gctattgc     60caaagacttt tgagaccgct tgctgtttca ttatagttga ggagttctcg aa#gacgagaa    120attagcagtt ttcggtgttt agtaatcgcg ctagcatgct aggacaattt aa#ctgcaaaa    180ttttgatacg atagtgatag taaatggaag gtaaaaataa catagaccta tc#aataagca    240atgtctctca gaataaaagc acttgatgca tcagtggtta acaaaattgc tg#caggtgag    300atcataatat cccccgtaaa tgctctcaaa gaaatgatgg agaattccat cg#atgcgaat    360gctacaatga ttgatattct agtcaaggaa ggaggaatta aggtacttca aa#taacagat    420aacggatctg gaattaataa agcagacctg ccaatcttat gtgagcgatt ca#cgacgtcc    480aaattacaaa aattcgaaga tttgagtcag attcaaacgt atggattccg ag#gagaagct    540ttagccagta tctcacatgt ggcaagagtc acagtaacga caaaagttaa ag#aagacaga    600tgtgcatgga gagtttcata tgcagaaggt aagatgttgg aaagccccaa ac#ctgttgct    660ggaaaagacg gtaccacgat cctagttgaa gacctttttt tcaatattcc tt#ctagatta    720agggccttga ggtcccataa tgatgaatac tctaaaatat tagatgttgt cg#ggcgatac    780gccattcatt ccaaggacat tggcttttct tgtaaaaagt tcggagactc ta#attattct    840ttatcagtta aaccttcata tacagtccag gataggatta ggactgtgtt ca#ataaatct    900gtggcttcga atttaattac ttttcatatc agcaaagtag aagatttaaa cc#tggaaagc    960gttgatggaa aggtgtgtaa tttgaatttc atatccaaaa agtccatttc at#taattttt   1020ttcattaata atagactagt gacatgtgat cttctaagaa gagctttgaa ca#gcgtttac   1080tccaattatc tgccaaaggg cttcagacct tttatttatt tgggaattgt ta#tagatccg   1140gcggctgttg atgttaacgt tcacccgaca aagagagagg ttcgtttcct ga#gccaagat   1200gagatcatag agaaaatcgc caatcaattg cacgccgaat tatctgccat tg#atacttca   1260cgtactttca aggcttcttc aatttcaaca aacaagccag agtcattgat ac#catttaat   1320gacaccatag aaagtgatag gaataggaag agtctccgac aagcccaagt gg#tagagaat   1380tcatatacga cagccaatag tcaactaagg aaagcgaaaa gacaagagaa ta#aactagtc   1440agaatagatg cttcacaagc taaaattacg tcatttttat cctcaagtca ac#agttcaac   1500tttgaaggat cgtctacaaa gcgacaactg agtgaaccca aggtaacaaa tg#taagccac   1560tcccaagagg cagaaaagct gacactaaat gaaagcgaac aaccgcgtga tg#ccaataca   1620atcaatgata atgacttgaa ggatcaacct aagaagaaac aaaagttggg gg#attataaa   1680gttccaagca ttgccgatga cgaaaagaat gcactcccga tttcaaaaga cg#ggtatatt   1740agagtaccta aggagcgagt taatgttaat cttacgagta tcaagaaatt gc#gtgaaaaa   1800gtagatgatt cgatacatcg agaactaaca gacatttttg caaatttgaa tt#acgttggg   1860gttgtagatg aggaaagaag attagccgct attcagcatg acttaaagct tt#ttttaata   1920gattacggat ctgtgtgcta tgagctattc tatcagattg gtttgacaga ct#tcgcaaac   1980tttggtaaga taaacctaca gagtacaaat gtgtcagatg atatagtttt gt#ataatctc   2040ctatcagaat ttgacgagtt aaatgacgat gcttccaaag aaaaaataat ta#gtaaaata   2100tgggacatga gcagtatgct aaatgagtac tattccatag aattggtgaa tg#atggtcta   2160gataatgact taaagtctgt gaagctaaaa tctctaccac tacttttaaa ag#gctacatt   2220ccatctctgg tcaagttacc attttttata tatcgcctgg gtaaagaagt tg#attgggag   2280gatgaacaag agtgtctaga tggtatttta agagagattg cattactcta ta#tacctgat   2340atggttccga aagtcgatac actcgatgca tcgttgtcag aagacgaaaa ag#cccagttt   2400ataaatagaa aggaacacat atcctcatta ctagaacacg ttctcttccc tt#gtatcaaa   2460cgaaggttcc tggcccctag acacattctc aaggatgtcg tggaaatagc ca#accttcca   2520gatctataca aagtttttga gaggtgttaa ctttaaaacg ttttggctgt aa#taccaaag   2580tttttgttta tttcctgagt gtgattgtgt ttcatttgaa agtgtatgcc ct#ttccttta   2640acgattcatc cgcgagattt caaaggatat gaaatatggt tgcagttagg aa#agtatgtc   2700agaaatgtat attcggattg aaactcttct aatagttctg aagtcacttg gt#tccgtatt   2760gttttcgtcc tcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gt#accaaaga   2820cccgagtcct tttatgagag aaaacatttc atcatttttc aactcaatta tc#ttaatatc   2880attttgtagt attttgaaaa caggatggta aaacgaatca cctgaatcta ga#agctgtac   2940cttgtcccat aaaagtttta atttactgag cctttcggtc aagtaaacta gt#ttatctag   3000ttttgaaccg aatattgtgg gcagatttgc agtaagttca gttagatcta ct#aaaagttg   3060tttgacagca gccgattcca caaaaatttg gtaaaaggag atgaaagaga cc#tcgcgcgt   3120aatggtttgc atcaccatcg gatgtctgtt gaaaaactca ctttttgcat gg#aagttatt   3180 aacaataaga ctaatgatta ccttagaata atgtataa      #                   #   3218 <210> SEQ ID NO 6 <211> LENGTH: 3056<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 6gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg ag#gagacaga     60taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ct#ggagacgc    120gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg ag#gagtcctg    180catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aa#gcctattg    240atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta ag#caccgctg    300tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ct#aaggctta    360aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta ga#agaagaaa    420actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gc#cgacctca    480cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gc#actaagtg    540atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gt#gtttgacc    600ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc ac#agtcagtg    660tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aa#cattaaaa    720aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gc#aggcgtcc    780gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tg#cacaagcg    840gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag tt#gcaaagcc    900tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac gg#cctgagca    960cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gc#acgcacgg   1020cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cc#tgtgaccc   1080agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg ca#tcagtacc   1140catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gt#aactccag   1200ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aa#gacctcct   1260tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag cc#actgctag   1320atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gt#gccaggaa   1380agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta gc#atccatct   1440ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct ag#gggtccag   1500agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tc#ctcttatc   1560cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gt#gagtccca   1620cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca gg#gctcagca   1680gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt ag#ctttagca   1740gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aa#ctgtggtg   1800acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac ca#tggatatc   1860aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc tt#caagacag   1920aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag ag#cacctcag   1980cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc ga#gttctctc   2040tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aa#caaacatg   2100aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gc#agcagaag   2160atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc tt#gggtcagt   2220ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg ga#ccagcatg   2280ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc ca#ggcgcaga   2340ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta ct#gatagaaa   2400atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gc#tccagtca   2460ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt gg#accccaag   2520atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc cg#gccctcac   2580gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt gg#aacggcgc   2640tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac ca#cccctgga   2700actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc at#ctctcaga   2760actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aa#agagaagg   2820ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gt#actggatc   2880catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca gc#tacttggg   2940tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc ca#cattcatg   3000agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aa#aaaa       3056 <210> SEQ ID NO 7 <211> LENGTH: 2771 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 7cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac ag#aacctgct     60aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gc#aggtggta    120ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tg#gtgccact    180aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc ag#acaatgga    240tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac at#ctaagatt    300caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga ag#ctctgagc    360tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc ga#aggttgga    420actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cc#cccgcccc    480agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg cc#ataaggaa    540tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tg#catactgt    600atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aa#aacgacag    660cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tg#tgtttggg    720cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ct#ccgtgtgt    780gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ct#caggtttc    840atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tt#tctttatc    900aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ct#accacatg    960tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc ag#aatgcgtt   1020gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gc#ttttgttg   1080gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gc#taaatgtc   1140agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc ag#cggatttg   1200gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg ag#aagaaaaa   1260aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aa#cagagaac   1320aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca ga#aaaggggt   1380atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag ac#ctcagaaa   1440gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga gg#tggagaag   1500gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc ag#acacgggc   1560agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gc#aggaacat   1620gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt gg#actgccat   1680tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac ta#atctcgca   1740accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga ca#tttgtcaa   1800aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt ga#aaattaat   1860aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aa#agcagtta   1920catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag gg#caaagatt   1980tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag ta#aaacgatg   2040tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac ca#aactgaat   2100gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cg#agatgctg   2160cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct ca#acttaact   2220gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tg#gctttgat   2280tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc ct#tgccaact   2340agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gc#tgagcgac   2400agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc ca#gagcctgc   2460cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa ac#tgatcacc   2520cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac ca#tgagacac   2580atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aa#taattggt   2640tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt tt#gttttaaa   2700atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tc#ttgagaac   2760 cttttcaaac c                #                  #                   #     2771 <210> SEQ ID NO 8 <211> LENGTH: 3063<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc ta#gcagcaag     60ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ct#cctttcaa    120gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aa#ctccttgg    180atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aa#aattgagg    240tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca at#gaagtact    300acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac gg#ttttcgtg    360gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca ag#aacggctg    420ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tc#tcagaaac    480cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aa#tctacctg    540taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aa#gatccaag    600atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gt#acataaca    660aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc at#gtcagttc    720tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa ga#atctcaga    780tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact ag#tctttcaa    840caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa ga#tatcttaa    900agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt tt#gtatcctg    960ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta ac#accagata   1020aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aa#tctgatga   1080cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa ac#agatgttt   1140ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aa#taaagtgg   1200aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc ca#aaatgata   1260tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag at#aagtattg   1320gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aa#cactaaga   1380atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag ac#ggaatata   1440gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aa#taaagacc   1500atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tc#ggaaattt   1560ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aa#tattgaac   1620ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aa#taattatc   1680caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aa#tgtaatag   1740ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta at#caagaaac   1800ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc at#agaaaatc   1860ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag ac#attgagtg   1920aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga ta#caatagtc   1980aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc ag#aaaaaaga   2040taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tc#attatcta   2100atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg ag#tcaaaata   2160ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aa#gaaacaaa   2220acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc ag#gtttcctg   2280atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat ag#agtagaag   2340aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag cc#actggaaa   2400agccaattat gttaacagag agtcttttta atggatctca ttatttagac gt#tttatata   2460aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cc#tcgtctta   2520cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aa#ttacttgg   2580aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aa#agaaattc   2640ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cg#caaagtga   2700taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg ta#cttatcaa   2760aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat ga#aattaaag   2820agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa ac#tacatgat   2880taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa ac#agcatgag   2940tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tg#aggattca   3000ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa at#aaactaat   3060 aac                   #                  #                   #           3063 <210> SEQ ID NO 9<211> LENGTH: 3145 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 9ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg ag#gtgaggag     60gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gc#cgaggtcg    120gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cg#ccttttcg    180accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cg#ggaggtgt    240tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat ct#gcagagtg    300ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gt#tcgtcagt    360atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aa#tgattggt    420atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ct#ctttggta    480acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gt#tgatggcc    540agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ct#gtgtgaat    600tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt gg#accaaagg    660aatgtgtttt acccggagga gagactgctg gagacatggg gaaactgaga ca#gataattc    720aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aa#agacattt    780atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat ag#tgctgtat    840tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aa#gtttttag    900aactcttatc agatgattcc aactttggac agtttgaact gactactttt ga#cttcagcc    960agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag gg#ttctgttg   1020aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa ac#ccctcaag   1080gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac ag#aatagagg   1140agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag ac#tttacaag   1200aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt ca#aagacaag   1260cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cc#taatgtta   1320tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca gt#ttttgtga   1380ctcctcttac tgatcttcgt tctgacttct ccaagtttca ggaaatgata ga#aacaactt   1440tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt ga#tcctaatc   1500tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca ac#attaataa   1560gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tc#cagtgcac   1620agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aa#caataaaa   1680actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc aa#attgactt   1740ctttaaatga agagtatacc aaaaataaaa cagaatatga agaagcccag ga#tgccattg   1800ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ct#caatgatg   1860tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gc#acctgttc   1920catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aa#agcatcca   1980ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gt#atactttg   2040aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aa#atcaacat   2100atattcgaca aactggggtg atagtactca tggcccaaat tgggtgtttt gt#gccatgtg   2160agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct gg#tgacagtc   2220aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct at#cctcaggt   2280ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tc#tacctacg   2340atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt gg#tgcttttt   2400gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata cc#aactgtta   2460ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt ta#tcaggtga   2520agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aa#tttcccta   2580agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt ca#gtatattg   2640gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ct#ggaaagag   2700agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg cc#ctttactg   2760aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gt#aatagcaa   2820agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tg#aaaaatcc   2880cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt at#attgtttt   2940atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca ac#ttaataag   3000atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aa#aaatgaga   3060gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gc#tgaatttt   3120 ataaataaaa tcatgtagtt tgtgg          #                   #             3145 <210> SEQ ID NO 10<211> LENGTH: 2484 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 10cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gc#tggacgag     60acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tg#ctatcaaa    120gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tg#ttaaagag    180ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa ag#aagatctg    240gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tt#tagccagt    300atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt gg#ctcatgtt    360actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ct#cagatgga    420aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat ca#cggtggag    480gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tg#aagaatat    540gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat ta#gtttctca    600gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ct#caaccgtg    660gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aa#ttggatgt    720gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ct#actcagtg    780aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac tt#ccttgaga    840aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc at#tcctgtac    900ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aa#agcatgaa    960gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cg#agagcaag   1020ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc ag#gacttgct   1080ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc ta#cttctgga   1140agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga ac#agaagctt   1200gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc ca#ttgtcaca   1260gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga ga#tgcttgaa   1320ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga ta#caacaaag   1380gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aa#agagacat   1440cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat ga#ctgcagct   1500tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca gg#aagaaatt   1560aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cg#tgggctgt   1620gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct ca#acaccacc   1680aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa tt#ttggtgtt   1740ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt ag#atagtcca   1800gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata ca#ttgttgag   1860tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tg#atgaggaa   1920gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt gg#agggactg   1980cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa gg#aatgtttt   2040gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta ca#tatctgag   2100gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ct#cctggaag   2160tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tc#ctaaacat   2220ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata ca#aagtcttt   2280gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ct#ctgtattc   2340cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gt#gttggtag   2400cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg at#tgattata   2460 aataaataga tgtgtcttaa cata          #                   #              2484 <210> SEQ ID NO 11<211> LENGTH: 426 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 11cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac ag#aacctgct     60aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gc#aggtggta    120ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tg#gtgccact    180aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc ag#acaatgga    240tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac at#ctaagatt    300caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga ag#ctctgagc    360tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc ga#aggttgga    420 acttga                  #                  #                   #          426 <210> SEQ ID NO 12 <211> LENGTH: 1408<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 12ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcct gc#cgtgcatg     60ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagac cc#ggtgacag    120ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggc gc#gtgccaga    180caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagac ac#caacgctc    240ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcgg aa#tccacgcc    300aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgtt cg#gaaaaaac    360gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcg ac#ctttgaca    420gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgccc gg#aaagggtg    480gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctg tg#ggagccgt    540ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttgga gc#gagctgag    600aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtc ag#tccatcag    660atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaagga gt#tagtagaa    720aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggacta tg#gagtggat    780ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga ag#gctttact    840ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggt gg#aaactttt    900ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcac ca#tttctacc    960tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgg ga#aaatcatc   1020cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagca gt#tattttct   1080acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacg tg#cctgcttc   1140cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccat gc#ttcctgta   1200cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctcctt gg#aggacaac   1260gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatg at#ctgcacaa   1320atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagt tc#ctgctgaa   1380 aatccaaaaa aaaaaaaaaa aaaaaaaa         #                   #           1408 <210> SEQ ID NO 13<211> LENGTH: 1785 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 13tttttagaaa ctgatgttta ttttccatca accatttttc catgctgctt aa#gagaatat     60gcaagaacag cttaagacca gtcagtggtt gctcctaccc attcagtggc ct#gagcagtg    120gggagctgca gaccagtctt ccgtggcagg ctgagcgctc cagtcttcag ta#gggaattg    180ctgaataggc acagagggca cctgtacacc ttcagaccag tctgcaacct ca#ggctgagt    240agcagtgaac tcaggagcgg gagcagtcca ttcaccctga aattcctcct tg#gtcactgc    300cttctcagca gcagcctgct cttctttttc aatctcttca ggatctctgt ag#aagtacag    360atcaggcatg acctcccatg ggtgttcacg ggaaatggtg ccacgcatgc gc#agaacttc    420ccgagccagc atccaccaca ttaaacccac tgagtgagct cccttgttgt tg#catgggat    480ggcaatgtcc acatagcgca gaggagaatc tgtgttacac agcgcaatgg ta#ggtaggtt    540aacataagat gcctccgtga gaggcgaagg ggcggcggga cccgggcctg gc#ccgtatgt    600gtccttggcg gcctagacta ggccgtcgct gtatggtgag ccccagggag gc#ggatctgg    660gcccccagaa ggacacccgc ctggatttgc cccgtagccc ggcccgggcc cc#tcgggagc    720agaacagcct tggtgaggtg gacaggaggg gacctcgcga gcagacgcgc gc#gccagcga    780cagcagcccc gccccggcct ctcgggagcc ggggggcaga ggctgcggag cc#ccaggagg    840gtctatcagc cacagtctct gcatgtttcc aagagcaaca ggaaatgaac ac#attgcagg    900ggccagtgtc attcaaagat gtggctgtgg atttcaccca ggaggagtgg cg#gcaactgg    960accctgatga gaagatagca tacggggatg tgatgttgga gaactacagc ca#tctagttt   1020ctgtggggta tgattatcac caagccaaac atcatcatgg agtggaggtg aa#ggaagtgg   1080agcagggaga ggagccgtgg ataatggaag gtgaatttcc atgtcaacat ag#tccagaac   1140ctgctaaggc catcaaacct attgatcgga agtcagtcca tcagatttgc tc#tgggccag   1200tggtactgag tctaagcact gcagtgaagg agttagtaga aaacagtctg ga#tgctggtg   1260ccactaatat tgatctaaag cttaaggact atggagtgga tctcattgaa gt#ttcagaca   1320atggatgtgg ggtagaagaa gaaaactttg aaggcttaat ctctttcagc tc#tgaaacat   1380cacacatgta agattcaaga gtttgccgac ctaactgaag ttgaaacttt cg#gttttcag   1440ggggaagctc tgagctcact gtgtgcactg agcgatgtca ccatttctac ct#gccacgcg   1500ttggtgaagg ttgggactcg actggtgttt gatcacgatg ggaaaatcat cc#aggaaacc   1560ccctaccccc accccagagg gaccacagtc agcgtgaagc agttattttc ta#cgctacct   1620gtgcgccata aggaatttca aaggaatatt aagaagacgt gcctgcttcc cc#ttcgcctt   1680ctgccgtgat tgtcagtttc ctgaggcctc cccagccatg cttcctgtac ag#cctgcaga   1740 actgtgagtc aattaaacct cttttcttca taaattaaaa aaaaa   #                1785 <210> SEQ ID NO 14 <211> LENGTH: 795<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 14atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag gg#aggcggat     60ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg gg#cccctcgg    120gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gc#gcgcgcca    180gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc gg#agccccag    240gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat ga#acacattg    300caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gt#ggcggcaa    360ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta ca#gccatcta    420gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga gg#tgaaggaa    480gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca ac#atagtcca    540gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat tt#gctctggg    600ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tc#tggatgct    660ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tg#aagtttca    720gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt ca#gctctgaa    780 acatcacaca tgtaa               #                  #                   #   795 <210> SEQ ID NO 15 <211> LENGTH: 769<212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 15 Met Ser Leu Arg Ile Lys Ala Leu Asp Ala Se#r Val Val Asn Lys Ile  1               5   #                10  #                15 Ala Ala Gly Glu Ile Ile Ile Ser Pro Val As#n Ala Leu Lys Glu Met             20       #            25      #            30 Met Glu Asn Ser Ile Asp Ala Asn Ala Thr Me#t Ile Asp Ile Leu Val         35           #        40          #        45 Lys Glu Gly Gly Ile Lys Val Leu Gln Ile Th#r Asp Asn Gly Ser Gly     50               #    55              #    60 Ile Asn Lys Ala Asp Leu Pro Ile Leu Cys Gl#u Arg Phe Thr Thr Ser 65                   #70                  #75                   #80 Lys Leu Gln Lys Phe Glu Asp Leu Ser Gln Il#e Gln Thr Tyr Gly Phe                 85   #                90  #                95 Arg Gly Glu Ala Leu Ala Ser Ile Ser His Va#l Ala Arg Val Thr Val             100       #           105      #           110 Thr Thr Lys Val Lys Glu Asp Arg Cys Ala Tr#p Arg Val Ser Tyr Ala         115           #       120          #       125 Glu Gly Lys Met Leu Glu Ser Pro Lys Pro Va#l Ala Gly Lys Asp Gly     130               #   135              #   140 Thr Thr Ile Leu Val Glu Asp Leu Phe Phe As#n Ile Pro Ser Arg Leu 145                 1 #50                 1#55                 1 #60 Arg Ala Leu Arg Ser His Asn Asp Glu Tyr Se#r Lys Ile Leu Asp Val                 165   #               170  #               175 Val Gly Arg Tyr Ala Ile His Ser Lys Asp Il#e Gly Phe Ser Cys Lys             180       #           185      #           190 Lys Phe Gly Asp Ser Asn Tyr Ser Leu Ser Va#l Lys Pro Ser Tyr Thr         195           #       200          #       205 Val Gln Asp Arg Ile Arg Thr Val Phe Asn Ly#s Ser Val Ala Ser Asn     210               #   215              #   220 Leu Ile Thr Phe His Ile Ser Lys Val Glu As#p Leu Asn Leu Glu Ser 225                 2 #30                 2#35                 2 #40 Val Asp Gly Lys Val Cys Asn Leu Asn Phe Il#e Ser Lys Lys Ser Ile                 245   #               250  #               255 Ser Leu Ile Phe Phe Ile Asn Asn Arg Leu Va#l Thr Cys Asp Leu Leu             260       #           265      #           270 Arg Arg Ala Leu Asn Ser Val Tyr Ser Asn Ty#r Leu Pro Lys Gly Phe         275           #       280          #       285 Arg Pro Phe Ile Tyr Leu Gly Ile Val Ile As#p Pro Ala Ala Val Asp     290               #   295              #   300 Val Asn Val His Pro Thr Lys Arg Glu Val Ar#g Phe Leu Ser Gln Asp 305                 3 #10                 3#15                 3 #20 Glu Ile Ile Glu Lys Ile Ala Asn Gln Leu Hi#s Ala Glu Leu Ser Ala                 325   #               330  #               335 Ile Asp Thr Ser Arg Thr Phe Lys Ala Ser Se#r Ile Ser Thr Asn Lys             340       #           345      #           350 Pro Glu Ser Leu Ile Pro Phe Asn Asp Thr Il#e Glu Ser Asp Arg Asn         355           #       360          #       365 Arg Lys Ser Leu Arg Gln Ala Gln Val Val Gl#u Asn Ser Tyr Thr Thr     370               #   375              #   380 Ala Asn Ser Gln Leu Arg Lys Ala Lys Arg Gl#n Glu Asn Lys Leu Val 385                 3 #90                 3#95                 4 #00 Arg Ile Asp Ala Ser Gln Ala Lys Ile Thr Se#r Phe Leu Ser Ser Ser                 405   #               410  #               415 Gln Gln Phe Asn Phe Glu Gly Ser Ser Thr Ly#s Arg Gln Leu Ser Glu             420       #           425      #           430 Pro Lys Val Thr Asn Val Ser His Ser Gln Gl#u Ala Glu Lys Leu Thr         435           #       440          #       445 Leu Asn Glu Ser Glu Gln Pro Arg Asp Ala As#n Thr Ile Asn Asp Asn     450               #   455              #   460 Asp Leu Lys Asp Gln Pro Lys Lys Lys Gln Ly#s Leu Gly Asp Tyr Lys 465                 4 #70                 4#75                 4 #80 Val Pro Ser Ile Ala Asp Asp Glu Lys Asn Al#a Leu Pro Ile Ser Lys                 485   #               490  #               495 Asp Gly Tyr Ile Arg Val Pro Lys Glu Arg Va#l Asn Val Asn Leu Thr             500       #           505      #           510 Ser Ile Lys Lys Leu Arg Glu Lys Val Asp As#p Ser Ile His Arg Glu         515           #       520          #       525 Leu Thr Asp Ile Phe Ala Asn Leu Asn Tyr Va#l Gly Val Val Asp Glu     530               #   535              #   540 Glu Arg Arg Leu Ala Ala Ile Gln His Asp Le#u Lys Leu Phe Leu Ile 545                 5 #50                 5#55                 5 #60 Asp Tyr Gly Ser Val Cys Tyr Glu Leu Phe Ty#r Gln Ile Gly Leu Thr                 565   #               570  #               575 Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gl#n Ser Thr Asn Val Ser             580       #           585      #           590 Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Gl#u Phe Asp Glu Leu Asn         595           #       600          #       605 Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Ly#s Ile Trp Asp Met Ser     610               #   615              #   620 Ser Met Leu Asn Glu Tyr Tyr Ser Ile Glu Le#u Val Asn Asp Gly Leu 625                 6 #30                 6#35                 6 #40 Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Se#r Leu Pro Leu Leu Leu                 645   #               650  #               655 Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pr#o Phe Phe Ile Tyr Arg             660       #           665      #           670 Leu Gly Lys Glu Val Asp Trp Glu Asp Glu Gl#n Glu Cys Leu Asp Gly         675           #       680          #       685 Ile Leu Arg Glu Ile Ala Leu Leu Tyr Ile Pr#o Asp Met Val Pro Lys     690               #   695              #   700 Val Asp Thr Leu Asp Ala Ser Leu Ser Glu As#p Glu Lys Ala Gln Phe 705                 7 #10                 7#15                 7 #20 Ile Asn Arg Lys Glu His Ile Ser Ser Leu Le#u Glu His Val Leu Phe                 725   #               730  #               735 Pro Cys Ile Lys Arg Arg Phe Leu Ala Pro Ar#g His Ile Leu Lys Asp             740       #           745      #           750 Val Val Glu Ile Ala Asn Leu Pro Asp Leu Ty#r Lys Val Phe Glu Arg         755           #       760          #       765 Cys <210> SEQ ID NO 16 <211> LENGTH: 859 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 16Met Glu Gln Thr Glu Gly Val Ser Thr Glu Cy #s Ala Lys Ala Ile Lys 1               5   #                10   #                15Pro Ile Asp Gly Lys Ser Val His Gln Ile Cy #s Ser Gly Gln Val Ile            20       #            25       #            30Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Il #e Glu Asn Ser Val Asp        35           #        40           #        45Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Ly #s Asp Tyr Gly Val Asp    50               #    55               #    60Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Va #l Glu Glu Glu Asn Phe65                   #70                   #75                   #80Glu Gly Leu Ala Leu Lys His His Thr Ser Ly #s Ile Gln Glu Phe Ala                85   #                90   #                95Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Ar #g Gly Glu Ala Leu Ser            100       #           105       #           110Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Se #r Thr Cys His Gly Ser        115           #       120           #       125Ala Ser Val Gly Thr Arg Leu Val Phe Asp Hi #s Asn Gly Lys Ile Thr    130               #   135               #   140Gln Lys Thr Pro Tyr Pro Arg Pro Lys Gly Th #r Thr Val Ser Val Gln145                 1 #50                 1 #55                 1 #60His Leu Phe Tyr Thr Leu Pro Val Arg Tyr Ly #s Glu Phe Gln Arg Asn                165   #               170   #               175Ile Lys Lys Glu Tyr Ser Lys Met Val Gln Va #l Leu Gln Ala Tyr Cys            180       #           185       #           190Ile Ile Ser Ala Gly Val Arg Val Ser Cys Th #r Asn Gln Leu Gly Gln        195           #       200           #       205Gly Lys Arg His Ala Val Val Cys Thr Ser Gl #y Thr Ser Gly Met Lys    210               #   215               #   220Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gl #n Leu Gln Ser Leu Ile225                 2 #30                 2 #35                 2 #40Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Va #l Cys Glu Glu Tyr Gly                245   #               250   #               255Leu Ser Thr Ser Gly Arg His Lys Thr Phe Se #r Thr Phe Arg Ala Ser            260       #           265       #           270Phe His Ser Ala Arg Thr Ala Pro Gly Gly Va #l Gln Gln Thr Gly Ser        275           #       280           #       285Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gl #n Gln Arg Ser Leu Ser    290               #   295               #   300Leu Ser Met Arg Phe Tyr His Met Tyr Asn Ar #g His Gln Tyr Pro Phe305                 3 #10                 3 #15                 3 #20Val Val Leu Asn Val Ser Val Asp Ser Glu Cy #s Val Asp Ile Asn Val                325   #               330   #               335Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Gl #u Glu Lys Leu Leu Leu            340       #           345       #           350Ala Val Leu Lys Thr Ser Leu Ile Gly Met Ph #e Asp Ser Asp Ala Asn        355           #       360           #       365Lys Leu Asn Val Asn Gln Gln Pro Leu Leu As #p Val Glu Gly Asn Leu    370               #   375               #   380Val Lys Leu His Thr Ala Glu Leu Glu Lys Pr #o Val Pro Gly Lys Gln385                 3 #90                 3 #95                 4 #00Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala As #p Glu Lys Arg Val Ala                405   #               410   #               415Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Le #u His Pro Thr Lys Glu            420       #           425       #           430Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Le #u Thr Arg Ser Phe Pro        435           #       440           #       445Ser Glu Lys Arg Gly Val Leu Ser Ser Tyr Pr #o Ser Asp Val Ile Ser    450               #   455               #   460Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Le #u Val Ser Pro Thr Asp465                 4 #70                 4 #75                 4 #80Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Il #e Glu Lys Asp Ser Gly                485   #               490   #               495Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Gl #u Phe Ser Thr Pro Glu            500       #           505       #           510Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Va #l Ser Ser Leu Glu Asp        515           #       520           #       525Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly As #p Leu Asp Cys Arg Pro    530               #   535               #   540Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu As #p His Gly Tyr Gln Cys545                 5 #50                 5 #55                 5 #60Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Th #r Asn Ala Lys Arg Phe                565   #               570   #               575Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Il #e Ser Gln Arg Leu Pro            580       #           585       #           590Gly Pro Gln Ser Thr Ser Ala Ala Glu Val As #p Val Ala Ile Lys Met        595           #       600           #       605Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Le #u Ser Ser Leu Ala Lys    610               #   615               #   620Arg Met Lys Gln Leu Gln His Leu Lys Ala Gl #n Asn Lys His Glu Leu625                 6 #30                 6 #35                 6 #40Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pr #o Gly Glu Asn Gln Ala                645   #               650   #               655Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Ly #s Ser Met Phe Ala Glu            660       #           665       #           670Met Glu Ile Leu Gly Gln Phe Asn Leu Gly Ph #e Ile Val Thr Lys Leu        675           #       680           #       685Lys Glu Asp Leu Phe Leu Val Asp Gln His Al #a Ala Asp Glu Lys Tyr    690               #   695               #   700Asn Phe Glu Met Leu Gln Gln His Thr Val Le #u Gln Ala Gln Arg Leu705                 7 #10                 7 #15                 7 #20Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Va #l Asn Glu Ala Val Leu                725   #               730   #               735Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gl #y Phe Asp Phe Val Ile            740       #           745       #           750Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Ly #s Leu Ile Ser Leu Pro        755           #       760           #       765Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln As #p Ile Asp Glu Leu Ile    770               #   775               #   780Phe Met Leu Ser Asp Ser Pro Gly Val Met Cy #s Arg Pro Ser Arg Val785                 7 #90                 7 #95                 8 #00Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Ly #s Ser Val Met Ile Gly                805   #               810   #               815Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Le #u Ile Thr His Met Gly            820       #           825       #           830Glu Met Asp His Pro Trp Asn Cys Pro His Gl #y Arg Pro Thr Met Arg        835           #       840           #       845His Val Ala Asn Leu Asp Val Ile Ser Gln As #n     850              #   855 <210> SEQ ID NO 17 <211> LENGTH: 932 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 17Met Lys Gln Leu Pro Ala Ala Thr Val Arg Le #u Leu Ser Ser Ser Gln 1               5   #                10   #                15Ile Ile Thr Ser Val Val Ser Val Val Lys Gl #u Leu Ile Glu Asn Ser            20       #            25       #            30Leu Asp Ala Gly Ala Thr Ser Val Asp Val Ly #s Leu Glu Asn Tyr Gly        35           #        40           #        45Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Gl #u Gly Ile Lys Ala Val    50               #    55               #    60Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Th #r Ser Lys Ile Asn Ser65                   #70                   #75                   #80His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gl #y Phe Arg Gly Glu Ala                85   #                90   #                95Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Le #u Ile Thr Thr Arg Thr            100       #           105       #           110Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Le #u Asp Gly Ser Gly His        115           #       120           #       125Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gl #n Gly Thr Thr Val Thr    130               #   135               #   140Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Ar #g Lys Gln Phe Tyr Ser145                 1 #50                 1 #55                 1 #60Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Ly #s Ile Gln Asp Leu Leu                165   #               170   #               175Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Ar #g Ile Val Phe Val His            180       #           185       #           190Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Va #l Ser Asp His Lys Met        195           #       200           #       205Ala Leu Met Ser Val Leu Gly Thr Ala Val Me #t Asn Asn Met Glu Ser    210               #   215               #   220Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Ty #r Leu Ser Gly Phe Leu225                 2 #30                 2 #35                 2 #40Pro Lys Cys Asp Ala Asp His Ser Phe Thr Se #r Leu Ser Thr Pro Glu                245   #               250   #               255Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Va #l His Gln Lys Asp Ile            260       #           265       #           270Leu Lys Leu Ile Arg His His Tyr Asn Leu Ly #s Cys Leu Lys Glu Ser        275           #       280           #       285Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Il #e Asp Val Pro Thr Ala    290               #   295               #   300Asp Val Asp Val Asn Leu Thr Pro Asp Lys Se #r Gln Val Leu Leu Gln305                 3 #10                 3 #15                 3 #20Asn Lys Glu Ser Val Leu Ile Ala Leu Glu As #n Leu Met Thr Thr Cys                325   #               330   #               335Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Gl #u Asn Asn Lys Thr Asp            340       #           345       #           350Val Ser Ala Ala Asp Ile Val Leu Ser Lys Th #r Ala Glu Thr Asp Val        355           #       360           #       365Leu Phe Asn Lys Val Glu Ser Ser Gly Lys As #n Tyr Ser Asn Val Asp    370               #   375               #   380Thr Ser Val Ile Pro Phe Gln Asn Asp Met Hi #s Asn Asp Glu Ser Gly385                 3 #90                 3 #95                 4 #00Lys Asn Thr Asp Asp Cys Leu Asn His Gln Il #e Ser Ile Gly Asp Phe                405   #               410   #               415Gly Tyr Gly His Cys Ser Ser Glu Ile Ser As #n Ile Asp Lys Asn Thr            420       #           425       #           430Lys Asn Ala Phe Gln Asp Ile Ser Met Ser As #n Val Ser Trp Glu Asn        435           #       440           #       445Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Il #e Ser Ser Val Lys His    450               #   455               #   460Thr Gln Ser Glu Asn Gly Asn Lys Asp His Il #e Asp Glu Ser Gly Glu465                 4 #70                 4 #75                 4 #80Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Se #r Glu Ile Ser Ala Asp                485   #               490   #               495Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Se #r Val Gly Glu Asn Ile            500       #           505       #           510Glu Pro Val Lys Ile Leu Val Pro Glu Lys Se #r Leu Pro Cys Lys Val        515           #       520           #       525Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Me #t Asn Leu Asn Glu Asp    530               #   535               #   540Ser Cys Asn Lys Lys Ser Asn Val Ile Asp As #n Lys Ser Gly Lys Val545                 5 #50                 5 #55                 5 #60Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Il #e Lys Lys Pro Met Ser                565   #               570   #               575Ala Ser Ala Leu Phe Val Gln Asp His Arg Pr #o Gln Phe Leu Ile Glu            580       #           585       #           590Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Le #u Gln Ile Glu Glu Leu        595           #       600           #       605Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Ly #s Tyr Glu Glu Lys Ala    610               #   615               #   620Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Me #t Lys Arg Ala Ile Glu625                 6 #30                 6 #35                 6 #40Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Ar #g Lys Lys Ile Lys Pro                645   #               650   #               655Thr Ser Ala Trp Asn Leu Ala Gln Lys His Ly #s Leu Lys Thr Ser Leu            660       #           665       #           670Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gl #n Ser Gln Ile Glu Lys        675           #       680           #       685Arg Arg Ser Gln Asn Ile Lys Met Val Gln Il #e Pro Phe Ser Met Lys    690               #   695               #   700Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Ly #s Val Asp Leu Glu Glu705                 7 #10                 7 #15                 7 #20Lys Asp Glu Pro Cys Leu Ile His Asn Leu Ar #g Phe Pro Asp Ala Trp                725   #               730   #               735Leu Met Thr Ser Lys Thr Glu Val Met Leu Le #u Asn Pro Tyr Arg Val            740       #           745       #           750Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Gl #u Asn His Lys Leu Pro        755           #       760           #       765Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Th #r Glu Ser Leu Phe Asn    770               #   775               #   780Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Me #t Thr Ala Asp Asp Gln785                 7 #90                 7 #95                 8 #00Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pr #o Arg Leu Thr Ala Asn                805   #               810   #               815Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Se #r Ile Thr Glu Asn Tyr            820       #           825       #           830Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pr #o Phe Tyr Gly Val Ala        835           #       840           #       845Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu As #n Arg Asn Ala Lys Glu    850               #   855               #   860Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Se #r Tyr Leu Glu Gly Glu865                 8 #70                 8 #75                 8 #80Ala Val Arg Leu Ser Arg Gln Leu Pro Met Ty #r Leu Ser Lys Glu Asp                885   #               890   #               895Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gl #n Phe Gly Asn Glu Ile            900       #           905       #           910Lys Glu Cys Val His Gly Arg Pro Phe Phe Hi #s His Leu Thr Tyr Leu        915           #       920           #       925 Pro Glu Thr Thr    930 <210> SEQ ID NO 18 <211> LENGTH: 932 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 18Met Lys Gln Leu Pro Ala Ala Thr Val Arg Le #u Leu Ser Ser Ser Gln 1               5   #                10   #                15Ile Ile Thr Ser Val Val Ser Val Val Lys Gl #u Leu Ile Glu Asn Ser            20       #            25       #            30Leu Asp Ala Gly Ala Thr Ser Val Asp Val Ly #s Leu Glu Asn Tyr Gly        35           #        40           #        45Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Gl #u Gly Ile Lys Ala Val    50               #    55               #    60Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Th #r Ser Lys Ile Asn Ser65                   #70                   #75                   #80His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gl #y Phe Arg Gly Glu Ala                85   #                90   #                95Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Le #u Ile Thr Thr Arg Thr            100       #           105       #           110Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Le #u Asp Gly Ser Gly His        115           #       120           #       125Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gl #n Gly Thr Thr Val Thr    130               #   135               #   140Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Ar #g Lys Gln Phe Tyr Ser145                 1 #50                 1 #55                 1 #60Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Ly #s Ile Gln Asp Leu Leu                165   #               170   #               175Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Ar #g Ile Val Phe Val His            180       #           185       #           190Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Va #l Ser Asp His Lys Met        195           #       200           #       205Ala Leu Met Ser Val Leu Gly Thr Ala Val Me #t Asn Asn Met Glu Ser    210               #   215               #   220Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Ty #r Leu Ser Gly Phe Leu225                 2 #30                 2 #35                 2 #40Pro Lys Cys Asp Ala Asp His Ser Phe Thr Se #r Leu Ser Thr Pro Glu                245   #               250   #               255Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Va #l His Gln Lys Asp Ile            260       #           265       #           270Leu Lys Leu Ile Arg His His Tyr Asn Leu Ly #s Cys Leu Lys Glu Ser        275           #       280           #       285Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Il #e Asp Val Pro Thr Ala    290               #   295               #   300Asp Val Asp Val Asn Leu Thr Pro Asp Lys Se #r Gln Val Leu Leu Gln305                 3 #10                 3 #15                 3 #20Asn Lys Glu Ser Val Leu Ile Ala Leu Glu As #n Leu Met Thr Thr Cys                325   #               330   #               335Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Gl #u Asn Asn Lys Thr Asp            340       #           345       #           350Val Ser Ala Ala Asp Ile Val Leu Ser Lys Th #r Ala Glu Thr Asp Val        355           #       360           #       365Leu Phe Asn Lys Val Glu Ser Ser Gly Lys As #n Tyr Ser Asn Val Asp    370               #   375               #   380Thr Ser Val Ile Pro Phe Gln Asn Asp Met Hi #s Asn Asp Glu Ser Gly385                 3 #90                 3 #95                 4 #00Lys Asn Thr Asp Asp Cys Leu Asn His Gln Il #e Ser Ile Gly Asp Phe                405   #               410   #               415Gly Tyr Gly His Cys Ser Ser Glu Ile Ser As #n Ile Asp Lys Asn Thr            420       #           425       #           430Lys Asn Ala Phe Gln Asp Ile Ser Met Ser As #n Val Ser Trp Glu Asn        435           #       440           #       445Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Il #e Ser Ser Val Lys His    450               #   455               #   460Thr Gln Ser Glu Asn Gly Asn Lys Asp His Il #e Asp Glu Ser Gly Glu465                 4 #70                 4 #75                 4 #80Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Se #r Glu Ile Ser Ala Asp                485   #               490   #               495Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Se #r Val Gly Glu Asn Ile            500       #           505       #           510Glu Pro Val Lys Ile Leu Val Pro Glu Lys Se #r Leu Pro Cys Lys Val        515           #       520           #       525Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Me #t Asn Leu Asn Glu Asp    530               #   535               #   540Ser Cys Asn Lys Lys Ser Asn Val Ile Asp As #n Lys Ser Gly Lys Val545                 5 #50                 5 #55                 5 #60Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Il #e Lys Lys Pro Met Ser                565   #               570   #               575Ala Ser Ala Leu Phe Val Gln Asp His Arg Pr #o Gln Phe Leu Ile Glu            580       #           585       #           590Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Le #u Gln Ile Glu Glu Leu        595           #       600           #       605Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Ly #s Tyr Glu Glu Lys Ala    610               #   615               #   620Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Me #t Lys Arg Ala Ile Glu625                 6 #30                 6 #35                 6 #40Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Ar #g Lys Lys Ile Lys Pro                645   #               650   #               655Thr Ser Ala Trp Asn Leu Ala Gln Lys His Ly #s Leu Lys Thr Ser Leu            660       #           665       #           670Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gl #n Ser Gln Ile Glu Lys        675           #       680           #       685Arg Arg Ser Gln Asn Ile Lys Met Val Gln Il #e Pro Phe Ser Met Lys    690               #   695               #   700Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Ly #s Val Asp Leu Glu Glu705                 7 #10                 7 #15                 7 #20Lys Asp Glu Pro Cys Leu Ile His Asn Leu Ar #g Phe Pro Asp Ala Trp                725   #               730   #               735Leu Met Thr Ser Lys Thr Glu Val Met Leu Le #u Asn Pro Tyr Arg Val            740       #           745       #           750Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Gl #u Asn His Lys Leu Pro        755           #       760           #       765Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Th #r Glu Ser Leu Phe Asn    770               #   775               #   780Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Me #t Thr Ala Asp Asp Gln785                 7 #90                 7 #95                 8 #00Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pr #o Arg Leu Thr Ala Asn                805   #               810   #               815Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Se #r Ile Thr Glu Asn Tyr            820       #           825       #           830Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pr #o Phe Tyr Gly Val Ala        835           #       840           #       845Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu As #n Arg Asn Ala Lys Glu    850               #   855               #   860Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Se #r Tyr Leu Glu Gly Glu865                 8 #70                 8 #75                 8 #80Ala Val Arg Leu Ser Arg Gln Leu Pro Met Ty #r Leu Ser Lys Glu Asp                885   #               890   #               895Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gl #n Phe Gly Asn Glu Ile            900       #           905       #           910Lys Glu Cys Val His Gly Arg Pro Phe Phe Hi #s His Leu Thr Tyr Leu        915           #       920           #       925 Pro Glu Thr Thr    930 <210> SEQ ID NO 19 <211> LENGTH: 934 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 19Met Ala Val Gln Pro Lys Glu Thr Leu Gln Le #u Glu Ser Ala Ala Glu 1               5   #                10   #                15Val Gly Phe Val Arg Phe Phe Gln Gly Met Pr #o Glu Lys Pro Thr Thr            20       #            25       #            30Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Ty #r Thr Ala His Gly Glu        35           #        40           #        45Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Ly #s Thr Gln Gly Val Ile    50               #    55               #    60Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Le #u Gln Ser Val Val Leu65                   #70                   #75                   #80Ser Lys Met Asn Phe Glu Ser Phe Val Lys As #p Leu Leu Leu Val Arg                85   #                90   #                95Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Al #a Gly Asn Lys Ala Ser            100       #           105       #           110Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys Al #a Ser Pro Gly Asn Leu        115           #       120           #       125Ser Gln Phe Glu Asp Ile Leu Phe Gly Asn As #n Asp Met Ser Ala Ser    130               #   135               #   140Ile Gly Val Val Gly Val Lys Met Ser Ala Va #l Asp Gly Gln Arg Gln145                 1 #50                 1 #55                 1 #60Val Gly Val Gly Tyr Val Asp Ser Ile Gln Ar #g Lys Leu Gly Leu Cys                165   #               170   #               175Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Le #u Glu Ala Leu Leu Ile            180       #           185       #           190Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gl #y Gly Glu Thr Ala Gly        195           #       200           #       205Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Ar #g Gly Gly Ile Leu Ile    210               #   215               #   220Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Ly #s Asp Ile Tyr Gln Asp225                 2 #30                 2 #35                 2 #40Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Gl #u Gln Met Asn Ser Ala                245   #               250   #               255Val Leu Pro Glu Met Glu Asn Gln Val Ala Va #l Ser Ser Leu Ser Ala            260       #           265       #           270Val Ile Lys Phe Leu Glu Leu Leu Ser Asp As #p Ser Asn Phe Gly Gln        275           #       280           #       285Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Ty #r Met Lys Leu Asp Ile    290               #   295               #   300Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gl #y Ser Val Glu Asp Thr305                 3 #10                 3 #15                 3 #20Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu As #n Lys Cys Lys Thr Pro                325   #               330   #               335Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Ly #s Gln Pro Leu Met Asp            340       #           345       #           350Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu Va #l Glu Ala Phe Val Glu        355           #       360           #       365Asp Ala Glu Leu Arg Gln Thr Leu Gln Glu As #p Leu Leu Arg Arg Phe    370               #   375               #   380Pro Asp Leu Asn Arg Leu Ala Lys Lys Phe Gl #n Arg Gln Ala Ala Asn385                 3 #90                 3 #95                 4 #00Leu Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Il #e Asn Gln Leu Pro Asn                405   #               410   #               415Val Ile Gln Ala Leu Glu Lys His Glu Gly Ly #s His Gln Lys Leu Leu            420       #           425       #           430Leu Ala Val Phe Val Thr Pro Leu Thr Asp Le #u Arg Ser Asp Phe Ser        435           #       440           #       445Lys Phe Gln Glu Met Ile Glu Thr Thr Leu As #p Met Asp Gln Val Glu    450               #   455               #   460Asn His Glu Phe Leu Val Lys Pro Ser Phe As #p Pro Asn Leu Ser Glu465                 4 #70                 4 #75                 4 #80Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Ly #s Met Gln Ser Thr Leu                485   #               490   #               495Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pr #o Gly Lys Gln Ile Lys            500       #           505       #           510Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Ph #e Arg Val Thr Cys Lys        515           #       520           #       525Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Ph #e Ser Thr Val Asp Ile    530               #   535               #   540Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Ly #s Leu Thr Ser Leu Asn545                 5 #50                 5 #55                 5 #60Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Gl #u Glu Ala Gln Asp Ala                565   #               570   #               575Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gl #y Tyr Val Glu Pro Met            580       #           585       #           590Gln Thr Leu Asn Asp Val Leu Ala Gln Leu As #p Ala Val Val Ser Phe        595           #       600           #       605Ala His Val Ser Asn Gly Ala Pro Val Pro Ty #r Val Arg Pro Ala Ile    610               #   615               #   620Leu Glu Lys Gly Gln Gly Arg Ile Ile Leu Ly #s Ala Ser Arg His Ala625                 6 #30                 6 #35                 6 #40Cys Val Glu Val Gln Asp Glu Ile Ala Phe Il #e Pro Asn Asp Val Tyr                645   #               650   #               655Phe Glu Lys Asp Lys Gln Met Phe His Ile Il #e Thr Gly Pro Asn Met            660       #           665       #           670Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gl #y Val Ile Val Leu Met        675           #       680           #       685Ala Gln Ile Gly Cys Phe Val Pro Cys Glu Se #r Ala Glu Val Ser Ile    690               #   695               #   700Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gl #y Asp Ser Gln Leu Lys705                 7 #10                 7 #15                 7 #20Gly Val Ser Thr Phe Met Ala Glu Met Leu Gl #u Thr Ala Ser Ile Leu                725   #               730   #               735Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Il #e Asp Glu Leu Gly Arg            740       #           745       #           750Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Al #a Trp Ala Ile Ser Glu        755           #       760           #       765Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Me #t Phe Ala Thr His Phe    770               #   775               #   780His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pr #o Thr Val Asn Asn Leu785                 7 #90                 7 #95                 8 #00His Val Thr Ala Leu Thr Thr Glu Glu Thr Le #u Thr Met Leu Tyr Gln                805   #               810   #               815Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gl #y Ile His Val Ala Glu            820       #           825       #           830Leu Ala Asn Phe Pro Lys His Val Ile Glu Cy #s Ala Lys Gln Lys Ala        835           #       840           #       845Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly Gl #u Ser Gln Gly Tyr Asp    850               #   855               #   860Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Le #u Glu Arg Glu Gln Gly865                 8 #70                 8 #75                 8 #80Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys Va #l Lys Gln Met Pro Phe                885   #               890   #               895Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Ly #s Leu Lys Gln Leu Lys            900       #           905       #           910Ala Glu Val Ile Ala Lys Asn Asn Ser Phe Va #l Asn Glu Ile Ile Ser        915           #       920           #       925Arg Ile Lys Val Thr Thr     930 <210> SEQ ID NO 20 <211> LENGTH: 756<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20Met Ser Phe Val Ala Gly Val Ile Arg Arg Le #u Asp Glu Thr Val Val 1               5   #                10   #                15Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Ar #g Pro Ala Asn Ala Ile            20       #            25       #            30Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Ly #s Ser Thr Ser Ile Gln        35           #        40           #        45Val Ile Val Lys Glu Gly Gly Leu Lys Leu Il #e Gln Ile Gln Asp Asn    50               #    55               #    60Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Il #e Val Cys Glu Arg Phe65                   #70                   #75                   #80Thr Thr Ser Lys Leu Gln Ser Phe Glu Asp Le #u Ala Ser Ile Ser Thr                85   #                90   #                95Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Il #e Ser His Val Ala His            100       #           105       #           110Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Ly #s Cys Ala Tyr Arg Ala        115           #       120           #       125Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pr #o Lys Pro Cys Ala Gly    130               #   135               #   140Asn Gln Gly Thr Gln Ile Thr Val Glu Asp Le #u Phe Tyr Asn Ile Ala145                 1 #50                 1 #55                 1 #60Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Gl #u Glu Tyr Gly Lys Ile                165   #               170   #               175Leu Glu Val Val Gly Arg Tyr Ser Val His As #n Ala Gly Ile Ser Phe            180       #           185       #           190Ser Val Lys Lys Gln Gly Glu Thr Val Ala As #p Val Arg Thr Leu Pro        195           #       200           #       205Asn Ala Ser Thr Val Asp Asn Ile Arg Ser Il #e Phe Gly Asn Ala Val    210               #   215               #   220Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu As #p Lys Thr Leu Ala Phe225                 2 #30                 2 #35                 2 #40Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Ty #r Ser Val Lys Lys Cys                245   #               250   #               255Ile Phe Leu Leu Phe Ile Asn His Arg Leu Va #l Glu Ser Thr Ser Leu            260       #           265       #           270Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Ty #r Leu Pro Lys Asn Thr        275           #       280           #       285His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Se #r Pro Gln Asn Val Asp    290               #   295               #   300Val Asn Val His Pro Thr Lys His Glu Val Hi #s Phe Leu His Glu Glu305                 3 #10                 3 #15                 3 #20Ser Ile Leu Glu Arg Val Gln Gln His Ile Gl #u Ser Lys Leu Leu Gly                325   #               330   #               335Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Th #r Leu Leu Pro Gly Leu            340       #           345       #           350Ala Gly Pro Ser Gly Glu Met Val Lys Ser Th #r Thr Ser Leu Thr Ser        355           #       360           #       365Ser Ser Thr Ser Gly Ser Ser Asp Lys Val Ty #r Ala His Gln Met Val    370               #   375               #   380Arg Thr Asp Ser Arg Glu Gln Lys Leu Asp Al #a Phe Leu Gln Pro Leu385                 3 #90                 3 #95                 4 #00Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Il #e Val Thr Glu Asp Lys                405   #               410   #               415Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gl #n Asp Glu Glu Met Leu            420       #           425       #           430Glu Leu Pro Ala Pro Ala Glu Val Ala Ala Ly #s Asn Gln Ser Leu Glu        435           #       440           #       445Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Se #r Glu Lys Arg Gly Pro    450               #   455               #   460Thr Ser Ser Asn Pro Arg Lys Arg His Arg Gl #u Asp Ser Asp Val Glu465                 4 #70                 4 #75                 4 #80Met Val Glu Asp Asp Ser Arg Lys Glu Met Th #r Ala Ala Cys Thr Pro                485   #               490   #               495Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Le #u Ser Leu Gln Glu Glu            500       #           505       #           510Ile Asn Glu Gln Gly His Glu Val Leu Arg Gl #u Met Leu His Asn His        515           #       520           #       525Ser Phe Val Gly Cys Val Asn Pro Gln Trp Al #a Leu Ala Gln His Gln    530               #   535               #   540Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Le #u Ser Glu Glu Leu Phe545                 5 #50                 5 #55                 5 #60Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn Ph #e Gly Val Leu Arg Leu                565   #               570   #               575Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Me #t Leu Ala Leu Asp Ser            580       #           585       #           590Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pr #o Lys Glu Gly Leu Ala        595           #       600           #       605Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Al #a Glu Met Leu Ala Asp    610               #   615               #   620Tyr Phe Ser Leu Glu Ile Asp Glu Glu Gly As #n Leu Ile Gly Leu Pro625                 6 #30                 6 #35                 6 #40Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Gl #u Gly Leu Pro Ile Phe                645   #               650   #               655Ile Leu Arg Leu Ala Thr Glu Val Asn Trp As #p Glu Glu Lys Glu Cys            660       #           665       #           670Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Ph #e Tyr Ser Ile Arg Lys        675           #       680           #       685Gln Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gl #y Gln Gln Ser Glu Val    690               #   695               #   700Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Th #r Val Glu His Ile Val705                 7 #10                 7 #15                 7 #20Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pr #o Lys His Phe Thr Glu                725   #               730   #               735Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pr #o Asp Leu Tyr Lys Val            740       #           745       #           750Phe Glu Arg Cys         755 <210> SEQ ID NO 21 <211> LENGTH: 133<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21Met Lys Gln Leu Pro Ala Ala Thr Val Arg Le #u Leu Ser Ser Ser Gln 1               5   #                10   #                15Ile Ile Thr Ser Val Val Ser Val Val Lys Gl #u Leu Ile Glu Asn Ser            20       #            25       #            30Leu Asp Ala Gly Ala Thr Ser Val Asp Val Ly #s Leu Glu Asn Tyr Gly        35           #        40           #        45Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Gl #u Gly Ile Lys Ala Val    50               #    55               #    60Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Th #r Ser Lys Ile Asn Ser65                   #70                   #75                   #80His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gl #y Phe Arg Gly Glu Ala                85   #                90   #                95Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Le #u Ile Thr Thr Arg Thr            100       #           105       #           110Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Le #u Asp Gly Ser Gly His        115           #       120           #       125Ile Leu Ser Gln Lys     130 <210> SEQ ID NO 22 <211> LENGTH: 1360<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22Met Ser Arg Gln Ser Thr Leu Tyr Ser Phe Ph #e Pro Lys Ser Pro Ala 1               5   #                10   #                15Leu Ser Asp Ala Asn Lys Ala Ser Ala Arg Al #a Ser Arg Glu Gly Gly            20       #            25       #            30Arg Ala Ala Ala Ala Pro Gly Ala Ser Pro Se #r Pro Gly Gly Asp Ala        35           #        40           #        45Ala Trp Ser Glu Ala Gly Pro Gly Pro Arg Pr #o Leu Ala Arg Ser Ala    50               #    55               #    60Ser Pro Pro Lys Ala Lys Asn Leu Asn Gly Gl #y Leu Arg Arg Ser Val65                   #70                   #75                   #80Ala Pro Ala Ala Pro Thr Ser Cys Asp Phe Se #r Pro Gly Asp Leu Val                85   #                90   #                95Trp Ala Lys Met Glu Gly Tyr Pro Trp Trp Pr #o Cys Leu Val Tyr Asn            100       #           105       #           110His Pro Phe Asp Gly Thr Phe Ile Arg Glu Ly #s Gly Lys Ser Val Arg        115           #       120           #       125Val His Val Gln Phe Phe Asp Asp Ser Pro Th #r Arg Gly Trp Val Ser    130               #   135               #   140Lys Arg Leu Leu Lys Pro Tyr Thr Gly Ser Ly #s Ser Lys Glu Ala Gln145                 1 #50                 1 #55                 1 #60Lys Gly Gly His Phe Tyr Ser Ala Lys Pro Gl #u Ile Leu Arg Ala Met                165   #               170   #               175Gln Arg Ala Asp Glu Ala Leu Asn Lys Asp Ly #s Ile Lys Arg Leu Glu            180       #           185       #           190Leu Ala Val Cys Asp Glu Pro Ser Glu Pro Gl #u Glu Glu Glu Glu Met        195           #       200           #       205Glu Val Gly Thr Thr Tyr Val Thr Asp Lys Se #r Glu Glu Asp Asn Glu    210               #   215               #   220Ile Glu Ser Glu Glu Glu Val Gln Pro Lys Th #r Gln Gly Ser Arg Arg225                 2 #30                 2 #35                 2 #40Ser Ser Arg Gln Ile Lys Lys Arg Arg Val Il #e Ser Asp Ser Glu Ser                245   #               250   #               255Asp Ile Gly Gly Ser Asp Val Glu Phe Lys Pr #o Asp Thr Lys Glu Glu            260       #           265       #           270Gly Ser Ser Asp Glu Ile Ser Ser Gly Val Gl #y Asp Ser Glu Ser Glu        275           #       280           #       285Gly Leu Asn Ser Pro Val Lys Val Ala Arg Ly #s Arg Lys Arg Met Val    290               #   295               #   300Thr Gly Asn Gly Ser Leu Lys Arg Lys Ser Se #r Arg Lys Glu Thr Pro305                 3 #10                 3 #15                 3 #20Ser Ala Thr Lys Gln Ala Thr Ser Ile Ser Se #r Glu Thr Lys Asn Thr                325   #               330   #               335Leu Arg Ala Phe Ser Ala Pro Gln Asn Ser Gl #u Ser Gln Ala His Val            340       #           345       #           350Ser Gly Gly Gly Asp Asp Ser Ser Arg Pro Th #r Val Trp Tyr His Glu        355           #       360           #       365Thr Leu Glu Trp Leu Lys Glu Glu Lys Arg Ar #g Asp Glu His Arg Arg    370               #   375               #   380Arg Pro Asp His Pro Asp Phe Asp Ala Ser Th #r Leu Tyr Val Pro Glu385                 3 #90                 3 #95                 4 #00Asp Phe Leu Asn Ser Cys Thr Pro Gly Met Ar #g Lys Trp Trp Gln Ile                405   #               410   #               415Lys Ser Gln Asn Phe Asp Leu Val Ile Cys Ty #r Lys Val Gly Lys Phe            420       #           425       #           430Tyr Glu Leu Tyr His Met Asp Ala Leu Ile Gl #y Val Ser Glu Leu Gly        435           #       440           #       445Leu Val Phe Met Lys Gly Asn Trp Ala His Se #r Gly Phe Pro Glu Ile    450               #   455               #   460Ala Phe Gly Arg Tyr Ser Asp Ser Leu Val Gl #n Lys Gly Tyr Lys Val465                 4 #70                 4 #75                 4 #80Ala Arg Val Glu Gln Thr Glu Thr Pro Glu Me #t Met Glu Ala Arg Cys                485   #               490   #               495Arg Lys Met Ala His Ile Ser Lys Tyr Asp Ar #g Val Val Arg Arg Glu            500       #           505       #           510Ile Cys Arg Ile Ile Thr Lys Gly Thr Gln Th #r Tyr Ser Val Leu Glu        515           #       520           #       525Gly Asp Pro Ser Glu Asn Tyr Ser Lys Tyr Le #u Leu Ser Leu Lys Glu    530               #   535               #   540Lys Glu Glu Asp Ser Ser Gly His Thr Arg Al #a Tyr Gly Val Cys Phe545                 5 #50                 5 #55                 5 #60Val Asp Thr Ser Leu Gly Lys Phe Phe Ile Gl #y Gln Phe Ser Asp Asp                565   #               570   #               575Arg His Cys Ser Arg Phe Arg Thr Leu Val Al #a His Tyr Pro Pro Val            580       #           585       #           590Gln Val Leu Phe Glu Lys Gly Asn Leu Ser Ly #s Glu Thr Lys Thr Ile        595           #       600           #       605Leu Lys Ser Ser Leu Ser Cys Ser Leu Gln Gl #u Gly Leu Ile Pro Gly    610               #   615               #   620Ser Gln Phe Trp Asp Ala Ser Lys Thr Leu Ar #g Thr Leu Leu Glu Glu625                 6 #30                 6 #35                 6 #40Glu Tyr Phe Arg Glu Lys Leu Ser Asp Gly Il #e Gly Val Met Leu Pro                645   #               650   #               655Gln Val Leu Lys Gly Met Thr Ser Glu Ser As #p Ser Ile Gly Leu Thr            660       #           665       #           670Pro Gly Glu Lys Ser Glu Leu Ala Leu Ser Al #a Leu Gly Gly Cys Val        675           #       680           #       685Phe Tyr Leu Lys Lys Cys Leu Ile Asp Gln Gl #u Leu Leu Ser Met Ala    690               #   695               #   700Asn Phe Glu Glu Tyr Ile Pro Leu Asp Ser As #p Thr Val Ser Thr Thr705                 7 #10                 7 #15                 7 #20Arg Ser Gly Ala Ile Phe Thr Lys Ala Tyr Gl #n Arg Met Val Leu Asp                725   #               730   #               735Ala Val Thr Leu Asn Asn Leu Glu Ile Phe Le #u Asn Gly Thr Asn Gly            740       #           745       #           750Ser Thr Glu Gly Thr Leu Leu Glu Arg Val As #p Thr Cys His Thr Pro        755           #       760           #       765Phe Gly Lys Arg Leu Leu Lys Gln Trp Leu Cy #s Ala Pro Leu Cys Asn    770               #   775               #   780His Tyr Ala Ile Asn Asp Arg Leu Asp Ala Il #e Glu Asp Leu Met Val785                 7 #90                 7 #95                 8 #00Val Pro Asp Lys Ile Ser Glu Val Val Glu Le #u Leu Lys Lys Leu Pro                805   #               810   #               815Asp Leu Glu Arg Leu Leu Ser Lys Ile His As #n Val Gly Ser Pro Leu            820       #           825       #           830Lys Ser Gln Asn His Pro Asp Ser Arg Ala Il #e Met Tyr Glu Glu Thr        835           #       840           #       845Thr Tyr Ser Lys Lys Lys Ile Ile Asp Phe Le #u Ser Ala Leu Glu Gly    850               #   855               #   860Phe Lys Val Met Cys Lys Ile Ile Gly Ile Me #t Glu Glu Val Ala Asp865                 8 #70                 8 #75                 8 #80Gly Phe Lys Ser Lys Ile Leu Lys Gln Val Il #e Ser Leu Gln Thr Lys                885   #               890   #               895Asn Pro Glu Gly Arg Phe Pro Asp Leu Thr Va #l Glu Leu Asn Arg Trp            900       #           905       #           910Asp Thr Ala Phe Asp His Glu Lys Ala Arg Ly #s Thr Gly Leu Ile Thr        915           #       920           #       925Pro Lys Ala Gly Phe Asp Ser Asp Tyr Asp Gl #n Ala Leu Ala Asp Ile    930               #   935               #   940Arg Glu Asn Glu Gln Ser Leu Leu Glu Tyr Le #u Glu Lys Gln Arg Asn945                 9 #50                 9 #55                 9 #60Arg Ile Gly Cys Arg Thr Ile Val Tyr Trp Gl #y Ile Gly Arg Asn Arg                965   #               970   #               975Tyr Gln Leu Glu Ile Pro Glu Asn Phe Thr Th #r Arg Asn Leu Pro Glu            980       #           985       #           990Glu Tyr Glu Leu Lys Ser Thr Lys Lys Gly Cy #s Lys Arg Tyr Trp Thr        995           #       1000           #      1005Lys Thr Ile Glu Lys Lys Leu Ala Asn Leu Il #e Asn Ala Glu Glu Arg    1010              #   1015               #  1020Arg Asp Val Ser Leu Lys Asp Cys Met Arg Ar #g Leu Phe Tyr Asn Phe1025                1030 #                1035  #               1040Asp Lys Asn Tyr Lys Asp Trp Gln Ser Ala Va #l Glu Cys Ile Ala Val                1045  #               1050   #              1055Leu Asp Val Leu Leu Cys Leu Ala Asn Tyr Se #r Arg Gly Gly Asp Gly            1060      #           1065       #          1070Pro Met Cys Arg Pro Val Ile Leu Leu Pro Gl #u Asp Thr Pro Pro Phe        1075          #       1080           #      1085Leu Glu Leu Lys Gly Ser Arg His Pro Cys Il #e Thr Lys Thr Phe Phe    1090              #   1095               #  1100Gly Asp Asp Phe Ile Pro Asn Asp Ile Leu Il #e Gly Cys Glu Glu Glu1105                1110 #                1115  #               1120Glu Gln Glu Asn Gly Lys Ala Tyr Cys Val Le #u Val Thr Gly Pro Asn                1125  #               1130   #              1135Met Gly Gly Lys Ser Thr Leu Met Arg Gln Al #a Gly Leu Leu Ala Val            1140      #           1145       #          1150Met Ala Gln Met Gly Cys Tyr Val Pro Ala Gl #u Val Cys Arg Leu Thr        1155          #       1160           #      1165Pro Ile Asp Arg Val Phe Thr Arg Leu Gly Al #a Ser Asp Arg Ile Met    1170              #   1175               #  1180Ser Gly Glu Ser Thr Phe Phe Val Glu Leu Se #r Glu Thr Ala Ser Ile1185                1190 #                1195  #               1200Leu Met His Ala Thr Ala His Ser Leu Val Le #u Val Asp Glu Leu Gly                1205  #               1210   #              1215Arg Gly Thr Ala Thr Phe Asp Gly Thr Ala Il #e Ala Asn Ala Val Val            1220      #           1225       #          1230Lys Glu Leu Ala Glu Thr Ile Lys Cys Arg Th #r Leu Phe Ser Thr His        1235          #       1240           #      1245Tyr His Ser Leu Val Glu Asp Tyr Ser Gln As #n Val Ala Val Arg Leu    1250              #   1255               #  1260Gly His Met Ala Cys Met Val Glu Asn Glu Cy #s Glu Asp Pro Ser Gln1265                1270 #                1275  #               1280Glu Thr Ile Thr Phe Leu Tyr Lys Phe Ile Ly #s Gly Ala Cys Pro Lys                1285  #               1290   #              1295Ser Tyr Gly Phe Asn Ala Ala Arg Leu Ala As #n Leu Pro Glu Glu Val            1300      #           1305       #          1310Ile Gln Lys Gly His Arg Lys Ala Arg Glu Ph #e Glu Lys Met Asn Gln        1315          #       1320           #      1325Ser Leu Arg Leu Phe Arg Glu Val Cys Leu Al #a Ser Glu Arg Ser Thr    1330              #   1335               #  1340Val Asp Ala Glu Ala Val His Lys Leu Leu Th #r Leu Ile Lys Glu Leu1345                1350 #                1355  #               1360<210> SEQ ID NO 23 <211> LENGTH: 389 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 23Met Ala Gln Pro Lys Gln Glu Arg Val Ala Ar #g Ala Arg His Gln Arg 1               5   #                10   #                15Ser Glu Thr Ala Arg His Gln Arg Ser Glu Th #r Ala Lys Thr Pro Thr            20       #            25       #            30Leu Gly Asn Arg Gln Thr Pro Thr Leu Gly As #n Arg Gln Thr Pro Arg        35           #        40           #        45Leu Gly Ile His Ala Arg Pro Arg Arg Arg Al #a Thr Thr Ser Leu Leu    50               #    55               #    60Thr Leu Leu Leu Ala Phe Gly Lys Asn Ala Va #l Arg Cys Ala Leu Ile65                   #70                   #75                   #80Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pr #o Leu Thr Glu Pro Leu                85   #                90   #                95Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pr #o Pro Arg Pro Glu Arg            100       #           105       #           110Val Glu His Asn Val Glu Ser Ser Arg Trp Gl #u Pro Arg Arg Arg Gly        115           #       120           #       125Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro Se #r Pro Arg Gly Gly Ser    130               #   135               #   140Gly Val Ala Ser Leu Glu Arg Ala Glu Asn Se #r Ser Thr Glu Pro Ala145                 1 #50                 1 #55                 1 #60Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Va #l His Gln Ile Cys Ser                165   #               170   #               175Gly Pro Val Val Pro Ser Leu Arg Pro Asn Al #a Val Lys Glu Leu Val            180       #           185       #           190Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Va #l Asp Leu Lys Leu Lys        195           #       200           #       205Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Gl #y Asn Gly Cys Gly Val    210               #   215               #   220Glu Glu Glu Asn Phe Glu Gly Phe Thr Leu Ly #s His His Thr Cys Lys225                 2 #30                 2 #35                 2 #40Ile Gln Glu Phe Ala Asp Leu Thr Gln Val Gl #u Thr Phe Gly Phe Arg                245   #               250   #               255Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Se #r Asp Val Thr Ile Ser            260       #           265       #           270Thr Cys Arg Val Ser Ala Lys Val Gly Thr Ar #g Leu Val Phe Asp His        275           #       280           #       285Tyr Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pr #o Arg Pro Arg Gly Met    290               #   295               #   300Thr Val Ser Val Lys Gln Leu Phe Ser Thr Le #u Pro Val His His Lys305                 3 #10                 3 #15                 3 #20Glu Phe Gln Arg Asn Ile Lys Lys Lys Arg Al #a Cys Phe Pro Phe Ala                325   #               330   #               335Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Se #r Pro Ala Met Leu Pro            340       #           345       #           350Val Gln Pro Val Glu Leu Thr Pro Arg Ser Th #r Pro Pro His Pro Cys        355           #       360           #       365Ser Leu Glu Asp Asn Val Ile Thr Val Phe Se #r Ser Val Lys Asn Gly    370               #   375               #   380 Pro Gly Ser Ser Arg385 <210> SEQ ID NO 24 <211> LENGTH: 264 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 24Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Ar #g Cys Met Val Ser Pro 1               5   #                10   #                15Arg Glu Ala Asp Leu Gly Pro Gln Lys Asp Th #r Arg Leu Asp Leu Pro            20       #            25       #            30Arg Ser Pro Ala Arg Ala Pro Arg Glu Gln As #n Ser Leu Gly Glu Val        35           #        40           #        45Asp Arg Arg Gly Pro Arg Glu Gln Thr Arg Al #a Pro Ala Thr Ala Ala    50               #    55               #    60Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Gl #u Ala Ala Glu Pro Gln65                   #70                   #75                   #80Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Ph #e Gln Glu Gln Gln Glu                85   #                90   #                95Met Asn Thr Leu Gln Gly Pro Val Ser Phe Ly #s Asp Val Ala Val Asp            100       #           105       #           110Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pr #o Asp Glu Lys Ile Ala        115           #       120           #       125Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser Hi #s Leu Val Ser Val Gly    130               #   135               #   140Tyr Asp Tyr His Gln Ala Lys His His His Gl #y Val Glu Val Lys Glu145                 1 #50                 1 #55                 1 #60Val Glu Gln Gly Glu Glu Pro Trp Ile Met Gl #u Gly Glu Phe Pro Cys                165   #               170   #               175Gln His Ser Pro Glu Pro Ala Lys Ala Ile Ly #s Pro Ile Asp Arg Lys            180       #           185       #           190Ser Val His Gln Ile Cys Ser Gly Pro Val Va #l Leu Ser Leu Ser Thr        195           #       200           #       205Ala Val Lys Glu Leu Val Glu Asn Ser Leu As #p Ala Gly Ala Thr Asn    210               #   215               #   220Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val As #p Leu Ile Glu Val Ser225                 2 #30                 2 #35                 2 #40Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Ph #e Glu Gly Leu Ile Ser                245   #               250   #               255Phe Ser Ser Glu Thr Ser His Met             260 <210> SEQ ID NO 25<211> LENGTH: 264 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 25 Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Ar#g Cys Met Val Ser Pro  1               5   #                10  #                15 Arg Glu Ala Asp Leu Gly Pro Gln Lys Asp Th#r Arg Leu Asp Leu Pro             20       #            25      #            30 Arg Ser Pro Ala Arg Ala Pro Arg Glu Gln As#n Ser Leu Gly Glu Val         35           #        40          #        45 Asp Arg Arg Gly Pro Arg Glu Gln Thr Arg Al#a Pro Ala Thr Ala Ala     50               #    55              #    60 Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Gl#u Ala Ala Glu Pro Gln 65                   #70                  #75                   #80 Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Ph#e Gln Glu Gln Gln Glu                 85   #                90  #                95 Met Asn Thr Leu Gln Gly Pro Val Ser Phe Ly#s Asp Val Ala Val Asp             100       #           105      #           110 Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pr#o Asp Glu Lys Ile Ala         115           #       120          #       125 Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser Hi#s Leu Val Ser Val Gly     130               #   135              #   140 Tyr Asp Tyr His Gln Ala Lys His His His Gl#y Val Glu Val Lys Glu 145                 1 #50                 1#55                 1 #60 Val Glu Gln Gly Glu Glu Pro Trp Ile Met Gl#u Gly Glu Phe Pro Cys                 165   #               170  #               175 Gln His Ser Pro Glu Pro Ala Lys Ala Ile Ly#s Pro Ile Asp Arg Lys             180       #           185      #           190 Ser Val His Gln Ile Cys Ser Gly Pro Val Va#l Leu Ser Leu Ser Thr         195           #       200          #       205 Ala Val Lys Glu Leu Val Glu Asn Ser Leu As#p Ala Gly Ala Thr Asn     210               #   215              #   220 Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val As#p Leu Ile Glu Val Ser 225                 2 #30                 2#35                 2 #40 Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Ph#e Glu Gly Leu Ile Ser                 245   #               250  #               255 Phe Ser Ser Glu Thr Ser His Met             260

We claim:
 1. A method for making a hypermutable yeast, comprising thestep of: introducing into a yeast a polynucleotide comprising a dominantnegative allele of a heterologous mismatch repair gene, wherein saidheterologous mismatch repair gene is a mammalian or plant MutL homolog,whereby the cell becomes hypermutable.
 2. The method of claim 1 whereinthe mismatch repair gene is PMS2.
 3. The method of claim 1 wherein themismatch repair gene is plant PMS2.
 4. The method of claim 1 wherein themismatch repair gene is MLH1.
 5. The method of claim 1 wherein themismatch repair gene is MLH3.
 6. The method of claim 1 wherein themismatch repair gene is a PMSR or PMSL homolog.
 7. The method of claim 2where the allele comprises a truncation mutation.
 8. The method of claim3 where the allele comprises a truncation mutation.
 9. The method ofclaim 2 wherein the allele comprises a truncation mutation at codon 134.10. The method of claim 1 wherein the polynucleotide is introduced intoa yeast by mating.
 11. The method of claim 2 wherein the mismatch repairgene is mammalian PMS2.
 12. The method of claim 8 wherein the mismatchrepair gene is plant PMS2.
 13. The method of claim 1 wherein themismatch repair gene is MLH1.
 14. The method of claim 1 wherein themismatch repair gene is MLH3.
 15. The method of claim 1 wherein themismatch repair gene is a plant MutL homolog.
 16. A homogeneouscomposition of cultured, hypermutable yeast comprising a dominantnegative allele of a mismatch repair gene, wherein said mismatch repairgene is a mammalian or plant MutL homolog.
 17. The homogeneouscomposition of cultured, hypermutable yeast of claim 16 wherein themismatch repair gene is a PMS2 gene or homolog.
 18. The homogeneouscomposition of cultured, hypermutable yeast of claim 16 wherein themismatch repair gene is a MLH1 or homolog thereof.
 19. The homogeneouscomposition of cultured, hypermutable yeast of claim 16 wherein themismatch repair gene is a PMSR homolog.
 20. The isolated hypermutableyeast of claim 17 wherein the cells express a protein consisting of thefirst 133 amino acids of PMS2.
 21. The isolated hypermutable yeast ofclaim 17 comprising a protein which consists of the first 133 aminoacids of PMS2.
 22. The isolated hypermutable yeast of claim 18comprising a protein which consists of a mammalian MutL protein.
 23. Amethod of generating a hypermutable cell comprising introducing into acell a polynucleotide encoding PMSR2, wherein said cell becomeshypermutable.
 24. The method of claim 23 wherein said cell is a yeastcell.
 25. The method of claim 24 wherein said yeast cell is of a genusselected from the group consisting of Saccharomyces,Schizosaccharomyces, Pichia, Hansenula, and Kluyverornyces.
 26. Themethod of claim 23 further comprising exposing said cell to a chemicalmutagen.
 27. A method for generating a mutation in a gene of interestcomprising the steps of growing a cell culture comprising the gene ofinterest and a polynucleotide encoding a PMSR2 operably linked toexpression control sequences; testing said cell to determine whether thegene of interest harbors a mutation.
 28. The method of claim 27 whereinthe step of testing comprises analyzing a nucleotide sequence of thegene of interest.
 29. The method of claim 27 wherein the step of testingcomprises analyzing mRNA transcribed from the gene of interest.
 30. Themethod of claim 27 wherein the step of testing comprises analyzing aprotein encoded by the gene of interest.
 31. The method of claim 27wherein the step of testing comprises analyzing the phenotype associatedwith the gene of interest.
 32. An isolated hypermutable cell comprisingan expression vector comprising a PMSR2 coding sequence operably linkedto expression control sequences.
 33. A method of generating ahypermutable cell comprising introducing into a cell a polynucleotideencoding PMSR3.
 34. The method of claim 33 wherein said cell is a yeastcell.
 35. The method of claim 34 wherein said yeast cell is of a genusselected from the group consisting of Saccharomyces,Schizosaccharomyces, Pichia, Hansenula, and Kluyveromyces.
 36. Themethod of claim 33 further comprising exposing said cell to a chemicalmutagen.
 37. A method for generating a mutation in a gene of interestcomprising the steps of: growing a cell culture comprising the gene ofinterest and a polynucleotide encoding a PMSR3 operably linked toexpression control sequences; testing said cell to determine whether thegene of interest harbors a mutation.
 38. The method of claim 37 whereinthe step of testing comprises analyzing a nucleotide sequence of thegene of interest.
 39. The method of claim 37 wherein the step of testingcomprises analyzing mRNA transcribed from the gene of interest.
 40. Themethod of claim 37 wherein the step of testing comprises analyzing aprotein encoded by the gene of interest.
 41. The method of claim 37wherein the step of testing comprises analyzing the phenotype associatedwith the gene of interest.
 42. An isolated hypermutable cell comprisingan expression vector comprising a PMSR3 coding sequence operably linkedto expression control sequences.